ERX1585495 E-MTAB-4870:C1HW8ACXX_13s002413_Pekowska_lane713s002413_ESC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234084 SAMEA4062974 C1HW8ACXX_13s002413_Pekowska_lane713s002413_ESC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 88 0 F Application Read Forward 1 1 R Application Read Reverse 45 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: embryonic stem cell cell type ERX1585496 E-MTAB-4870:C1HW8ACXX_13s002413_Pekowska_lane713s002413_ESC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234085 SAMEA4062975 C1HW8ACXX_13s002413_Pekowska_lane713s002413_ESC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 88 0 F Application Read Forward 1 1 R Application Read Reverse 45 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: embryonic stem cell cell type ERX1585497 E-MTAB-4870:C1HW8ACXX_13s002413_Pekowska_lane713s002413_NSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234086 SAMEA4062976 C1HW8ACXX_13s002413_Pekowska_lane713s002413_NSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 88 0 F Application Read Forward 1 1 R Application Read Reverse 45 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: neural stem cell cell type ERX1585498 E-MTAB-4870:C1HW8ACXX_13s002413_Pekowska_lane713s002413_NSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234087 SAMEA4062977 C1HW8ACXX_13s002413_Pekowska_lane713s002413_NSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 88 0 F Application Read Forward 1 1 R Application Read Reverse 45 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: neural stem cell cell type ERX1585499 E-MTAB-4870:C1HW8ACXX_13s002413_Pekowska_lane813s002413_ESC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234088 SAMEA4062978 C1HW8ACXX_13s002413_Pekowska_lane813s002413_ESC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 88 0 F Application Read Forward 1 1 R Application Read Reverse 45 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: embryonic stem cell cell type ERX1585500 E-MTAB-4870:C1HW8ACXX_13s002413_Pekowska_lane813s002413_ESC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234089 SAMEA4062979 C1HW8ACXX_13s002413_Pekowska_lane813s002413_ESC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 88 0 F Application Read Forward 1 1 R Application Read Reverse 45 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: embryonic stem cell cell type ERX1585501 E-MTAB-4870:C1HW8ACXX_13s002413_Pekowska_lane813s002413_NSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234090 SAMEA4062980 C1HW8ACXX_13s002413_Pekowska_lane813s002413_NSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 88 0 F Application Read Forward 1 1 R Application Read Reverse 45 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: neural stem cell cell type ERX1585502 E-MTAB-4870:C1HW8ACXX_13s002413_Pekowska_lane813s002413_NSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234091 SAMEA4062981 C1HW8ACXX_13s002413_Pekowska_lane813s002413_NSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 88 0 F Application Read Forward 1 1 R Application Read Reverse 45 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: neural stem cell cell type ERX1585503 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane512s008032_ESC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234092 SAMEA4062982 C1JDTACXX_12s008032_Pekowska_lane512s008032_ESC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: embryonic stem cell cell type ERX1585504 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane512s008032_ESC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234093 SAMEA4062983 C1JDTACXX_12s008032_Pekowska_lane512s008032_ESC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: embryonic stem cell cell type ERX1585505 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane512s008032_NSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234094 SAMEA4062984 C1JDTACXX_12s008032_Pekowska_lane512s008032_NSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: neural stem cell cell type ERX1585506 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane512s008032_NSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234095 SAMEA4062985 C1JDTACXX_12s008032_Pekowska_lane512s008032_NSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: neural stem cell cell type ERX1585507 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane612s008032_ESC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234096 SAMEA4062986 C1JDTACXX_12s008032_Pekowska_lane612s008032_ESC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: embryonic stem cell cell type ERX1585508 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane612s008032_ESC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234097 SAMEA4062987 C1JDTACXX_12s008032_Pekowska_lane612s008032_ESC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: embryonic stem cell cell type ERX1585509 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane612s008032_NSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234098 SAMEA4062988 C1JDTACXX_12s008032_Pekowska_lane612s008032_NSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: neural stem cell cell type ERX1585510 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane612s008032_NSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234099 SAMEA4062989 C1JDTACXX_12s008032_Pekowska_lane612s008032_NSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: neural stem cell cell type ERX1585511 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane712s008032_ESC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234100 SAMEA4062990 C1JDTACXX_12s008032_Pekowska_lane712s008032_ESC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: embryonic stem cell cell type ERX1585512 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane712s008032_ESC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234101 SAMEA4062991 C1JDTACXX_12s008032_Pekowska_lane712s008032_ESC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: embryonic stem cell cell type ERX1585513 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane712s008032_NSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234102 SAMEA4062992 C1JDTACXX_12s008032_Pekowska_lane712s008032_NSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: neural stem cell cell type ERX1585514 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane712s008032_NSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234103 SAMEA4062993 C1JDTACXX_12s008032_Pekowska_lane712s008032_NSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: neural stem cell cell type ERX1585515 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane812s008032_ESC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234104 SAMEA4062994 C1JDTACXX_12s008032_Pekowska_lane812s008032_ESC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: embryonic stem cell cell type ERX1585516 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane812s008032_ESC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234105 SAMEA4062995 C1JDTACXX_12s008032_Pekowska_lane812s008032_ESC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: embryonic stem cell cell type ERX1585517 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane812s008032_NSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234106 SAMEA4062996 C1JDTACXX_12s008032_Pekowska_lane812s008032_NSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: neural stem cell cell type ERX1585518 E-MTAB-4870:C1JDTACXX_12s008032_Pekowska_lane812s008032_NSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234107 SAMEA4062997 C1JDTACXX_12s008032_Pekowska_lane812s008032_NSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: neural stem cell cell type ERX1585519 E-MTAB-4870:C34M8ACXX_15s009462_Pekowska_lane215s009462_ESC1_2i_p ERP016348 TCC in pluripotent and differentiated cells ERS1234108 SAMEA4062998 C34M8ACXX_15s009462_Pekowska_lane215s009462_ESC1_2i_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: embryonic stem cell cell type ERX1585520 E-MTAB-4870:C34M8ACXX_15s009463_Pekowska_lane315s009463_ESC2_2i_p ERP016348 TCC in pluripotent and differentiated cells ERS1234109 SAMEA4062999 C34M8ACXX_15s009463_Pekowska_lane315s009463_ESC2_2i_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: embryonic stem cell cell type ERX1585521 E-MTAB-4870:C799JACXX_15s013537_Pekowska_EpiSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234110 SAMEA4063000 C799JACXX_15s013537_Pekowska_EpiSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 100 0 F Application Read Forward 1 1 R Application Read Reverse 51 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: epiblast-derived stem cell cell type ERX1585522 E-MTAB-4870:C799JACXX_15s013538_Pekowska_EpiSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234111 SAMEA4063001 C799JACXX_15s013538_Pekowska_EpiSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 100 0 F Application Read Forward 1 1 R Application Read Reverse 51 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: epiblast-derived stem cell cell type ERX1585523 E-MTAB-4870:C7AP3ACXX_15s012980_Pekowska_lane415s012980_EpiSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234112 SAMEA4063002 C7AP3ACXX_15s012980_Pekowska_lane415s012980_EpiSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: epiblast-derived stem cell cell type ERX1585524 E-MTAB-4870:C7AP3ACXX_15s012980_Pekowska_lane515s012981_EpiSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234113 SAMEA4063003 C7AP3ACXX_15s012980_Pekowska_lane515s012981_EpiSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 102 0 F Application Read Forward 1 1 R Application Read Reverse 52 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: epiblast-derived stem cell cell type ERX1585526 E-MTAB-4870:D179FACXX_12s006436_Pekowska_lane812s006436_ESC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234115 SAMEA4063005 D179FACXX_12s006436_Pekowska_lane812s006436_ESC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 196 0 F Application Read Forward 1 1 R Application Read Reverse 99 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: embryonic stem cell cell type ERX1585527 E-MTAB-4870:D179FACXX_12s006436_Pekowska_lane812s006436_ESC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234116 SAMEA4063006 D179FACXX_12s006436_Pekowska_lane812s006436_ESC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 192 0 F Application Read Forward 1 1 R Application Read Reverse 97 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: embryonic stem cell cell type ERX1585528 E-MTAB-4870:D179FACXX_12s006436_Pekowska_lane812s006436_NSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234117 SAMEA4063007 D179FACXX_12s006436_Pekowska_lane812s006436_NSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 196 0 F Application Read Forward 1 1 R Application Read Reverse 99 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: neural stem cell cell type ERX1585529 E-MTAB-4870:D179FACXX_12s006436_Pekowska_lane812s006436_NSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234118 SAMEA4063008 D179FACXX_12s006436_Pekowska_lane812s006436_NSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 192 0 F Application Read Forward 1 1 R Application Read Reverse 97 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: neural stem cell cell type ERX1585530 E-MTAB-4870:D1GUAACXX_12s007362_Pekowska_lane212s007362_ESC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234119 SAMEA4063009 D1GUAACXX_12s007362_Pekowska_lane212s007362_ESC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 196 0 F Application Read Forward 1 1 R Application Read Reverse 99 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: embryonic stem cell cell type ERX1585531 E-MTAB-4870:D1GUAACXX_12s007362_Pekowska_lane212s007362_ESC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234120 SAMEA4063010 D1GUAACXX_12s007362_Pekowska_lane212s007362_ESC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 196 0 F Application Read Forward 1 1 R Application Read Reverse 99 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: embryonic stem cell cell type ERX1585532 E-MTAB-4870:D1GUAACXX_12s007362_Pekowska_lane212s007362_NSC1_p ERP016348 TCC in pluripotent and differentiated cells ERS1234121 SAMEA4063011 D1GUAACXX_12s007362_Pekowska_lane212s007362_NSC1_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 196 0 F Application Read Forward 1 1 R Application Read Reverse 99 Illumina HiSeq 2000 Experimental Factor: biological replicate1 replicate Experimental Factor: neural stem cell cell type ERX1585533 E-MTAB-4870:D1GUAACXX_12s007362_Pekowska_lane212s007362_NSC2_p ERP016348 TCC in pluripotent and differentiated cells ERS1234122 SAMEA4063012 D1GUAACXX_12s007362_Pekowska_lane212s007362_NSC2_p WGS GENOMIC RANDOM Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. Buffers for the procedure, Lysis buffer: 10 mM HEPES pH=8, 10 mM NaCl, 0.2percent NP-40, Wash buffer: 50 mM Tris-HCl pH = 8, 50 mM NaCl, 1 mM EDTA, EZ-Link Iodoacetyl PEG2 Biotin resuspension buffer - EZ RB: 50 mM Tris-HCl, 5 mM EDTA, pH = 8 - 8.3, Dialysis buffer: 10 mM Tris HCl pH = 8, 1 mM EDTA, PBST: PBS, 0.01 percent of Tween, Beads wash buffer 10 mM Tris-HCl pH = 8, 50 mM NaCl, 0.4 percent Triton X-100, Klenow Wash Buffer: 50 mM Tris-HCl pH = 7.4, 0.4 percent Triton X-100, 0.1 mM EDTA, DNA extraction buffer: 50 mM Tris-HCl pH = 8, 0.2 percent SDS, 1 mM EDTA, 100 mM NaCl, DNA resuspension solution: 10 mM Tris-HCl pH = 8, Bind and Wash Buffer: 2x : 10 mM Tris-HCl pH = 7.5, 1mM EDTA, 2 M NaCl, Cell crossling protocol: Put 100 million cell pellet in 45 ml of fresh medium and add 1.25 ml of 37 percent formaldehyde, Incubate 10 min at RT, Add 2.5 ml of 2.5M glycine , Incubate 5 min at RT, Incubate 15 min on ice, Divide the cell mixture into 4 equal parts, Centrifuge at 1500 rpm for 5 min. at 4 degrees and store the pellets if needed. To start the TCC procedure, Lyse the cells in 550 ul lysis buffer completed with PIC using a homogenizer , Incubate on ice at least 15 minutes , Lyse the cells using the Dounce homogenizer by moving the pestile A up and down 10 times, Incubate on ice for 1 minutes , Apply 10 more strokes of the pestile, Transfer the sample to 2ml tube, Wash the cells twice with the wash buffer, spin conditions: 2500g for 5 minutes, V = 250 ul, Re suspend the cell pellet in 250 ul of wash buffer, Add 95 ul of 2percent SDS and incubate at 65 degreeC for 10 min, Prepare the EZ link Iodoacetyl-PEG2-Biotin For two reactions, Equilibrate the powder to RT to reduce humidification of the reagen, take 3 mg of the EZ link Iodoacetyl-PEG2-Biotin and re suspend in 220 ul of EZ RB in an amber tube, Cool the chromatin suspension to RT, Mix the suspension with 105ul of 25 mM EZlink Iodoacetyl-PEG2-Biotin (IPB), Rock at the room temperature for 1 hour, protect from light, Mix the chromatin with 1300 ul of 1x NEB Buffer 2, Incubate on ice for 5 minutes, Mix the sample with 225 ul of 10 percent triton X-100, Incubate on ice for 10 min , Incubate at 37degree C for 10 minutes, Now the sample is ready to be digested to this end, add, 100 ul of 10x NEB buffer 2, 350 ul of water, 5 ul of 1M DTT, 20 ul of HindIIII (100U/ul), dispose the sample to two tubes and incubate the digestion reaction OVN at 37 degreeC with overhead shaking.Place the mixture in a 20 kD cutoff Slide-A-Lyser Cassette, dialyse it against 1L of dialysis buffer for 3 hours, Renew the buffer and dialyze for an additional hour, Transfer the dialyzed sample (ca 2.5 ml) to a 15 ml falcon tube, During the last step, prepare the T1 beads. Take 400 ul of beads per chromatin sample, Wash the beads 3 times with PBST, Re suspend the beads in 2 ml of PBST, Divide the chromatin after dialysis into 5 equal parts, put them into 1.5 ml eppendorf tubes and add 400 ul of the above-mentioned bead suspension to these 5 aliquots of chromatin, Rock the chromatin with beads at RT for 30 minutes, During this time prepare the neutralized IPB solution - nIPB, take 0.750 mg IPB and add 52.5 ul of the resuspension buffer , add 7.75 mili-mols of bME - 9 ul of bME stock (14.3 Molar solution), Mix each aliquot of chromatin with 5 ul of nIPB, Incubate with rotation for 15 minutes at RT, Wash the beads once with 600 ul of TPBS, Wash the beads with 600 ul of Beads Wash Buffer, Re-suspend the beads in 100 ul of Beads Wash Buffer, add 69 ul of water, 1.1 ul of 1M MgCl2, 11 ul of 10x NEB buffer 2, 0.77 ul of 10 uM dATP, 0.77 ul of 10 uM dTTP, 0.77ul of 10 uM dGTPαS, 16.5 ul 0.4 mM Biotin-14-dCTP, 4.4 ul of 10 percent Triton-X100, 5 ul of 5U/ul Klenow. Fill in the overhangs by 40 minute rocking incubation at RT with 100 ul of the above-mentioned mix, After the incubation with Klenow add 5ul of 0.5 M EDTA to the suspension, Wash the beads twice with 600 ul of Klenow Wash Buffer, Re-suspend the beads in 500 ul of Klenow wash buffer and transfer them all in a new 50 ml falcon tube (V = 2.5 ml), Prepare the ligation mix (0.275 ul 10X NEB T4 Ligase buffer, 4.3 ml water, 198 ul 10percent triton X-100, 110 ul 1 M Tris-Hcl, pH=7.4, 55ul 10mg/ml BSA, 2ul Qucik Ligase NEB), Rock the mix for 4 hours at 16 degreeC , Stop the ligation by adding 200 ul of 0.5 M EDTA, Place the bead suspension on the magnet and aspirate the supernatant, Reverse the crosslinks: resuspend the beads in 400 ul of an extraction buffer, Add 20 ul of 20 mg/ml proteinase K, Incubate at 65 degreeC OVN , Add 5 ul of 20 mg/ml proteinase K, Incubate for 2 additional hours at 65 degreeC, Transfer the supernatant to a new tube and extract the DNA twice with, phenol : chloroform : isoamyl alcohol, Once with an equal volume of chloroform, Mix the resulting aqueous phase with , NaCl (final concentration: 200 mM), Glycogen (final concentration: 25 ug/ul), Add 900 ul of 98 percent ethanol, Incubate at -20 ovn or at -80 for 1 hour, Centrifuge for 20 minutes at 4 degreeC at max speed, Wash the pellet with 80 percent ethanol, Resuspend the pellet in 20 ul of DNA resuspension solution and pull the 5 samples, Mix the solution with 1 ul of RNase A and incubate for 30 minutes at 37 degreeC Isolate the DNA by phenol chloroform isolation and ethnol precipitation Resuspend the pellet in water. ExoIII treatment: take 5 ug of the purified DNA and resuspend it in a total volume of 90 ul of 1x NEB buffer 1 Add 300 U of ExoIII, Incubate for 1 hour at 37 degreeC, Stop the reaction by adding 2 ul of 0.5 M EDTA and 2 ul of 5 M NaCl, Incubate the reaction at 70 degreeC for 20 minutes, Add water to 100 ul and shear on Covaris S2 apparatus to an average size of 100-400 bp:, duty cycle: 5percent, intensity: 5, cycles/burst: 200, time: 180 seconds, Purify the DNA by Ampure bead solution, elute in 90 ul of water, End repair the sample for 30 minutes at 20 degrees: add 10 ul of 10x T4 ligase buffer supplemented with ATP and DTT, add 1.6 ul of 25 mM dNTP mix, 5 U of Klenow - 1ul, 15 U T4 polymerase - 5 ul, 50 U of PNK - 5ul, Purify the DNA by Quiaquick/Ampure beads, elute in 44 ul of water Add the A-overhangs incubate for 30 minutes at 37 degrees with :, add 5 ul of water, 5 ul of 10x NEB Buffer 2, 1 ul of 10 mM dATP solution , 3 ul of exo-Klenow, Stop the reaction by adding 1 ul of 0.5 EDTA, Wash 50 ul of MyOne Streptavidin C1 beads twice with 500 ul of 1x Wash and Bind buffer in DNA Lo-Bind tubes, Re suspend the beads in 50 ul of 2x, Wash and bind buffer, Mix the beads with the A-added DNA sample and rock the mixture for 30 min at RT, After the incubation, wash the beads once with 500 ul of supplemented bind Wash buffer., Wash the beads once in DNA resuspension solution, Now we would like to add sequencing adapters to this end re-suspend the beads in the following mix: 50 ul 2x rapid ligation mix, 3 ul of sequencing adapters (5uM), 47 ul of water, add 1 ul of rapid ligase, incubate at RT with mixing for 20 minutes, Stop the reaction by adding 6 ul of 0.5 M EDTA/100 ul of ligation mix, Wash the beads twice with 1x Bind and wash buffer, Wash the beads once with TE, Resuspend the beads in 30 ul of water Perform the PCR amplification and size excision. Prepare Phusion mix: 0.5 ul of primer, 5 ul of beads, 25 ul of Phusion MM, 19.5 ul of water. 196 0 F Application Read Forward 1 1 R Application Read Reverse 99 Illumina HiSeq 2000 Experimental Factor: biological replicate2 replicate Experimental Factor: neural stem cell cell type