SRX5566262 GSM3684264 SRP189280 PRJNA528662 SRS4529457 SAMN11239140 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684264 GSM3684264 GEO Accession GSM3684264 SRX5566263 GSM3684265 SRP189280 PRJNA528662 SRS4529458 SAMN11239139 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684265 GSM3684265 GEO Accession GSM3684265 SRX5566264 GSM3684266 SRP189280 PRJNA528662 SRS4529459 SAMN11239138 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684266 GSM3684266 GEO Accession GSM3684266 SRX5566265 GSM3684267 SRP189280 PRJNA528662 SRS4529460 SAMN11239137 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684267 GSM3684267 GEO Accession GSM3684267 SRX5566266 GSM3684268 SRP189280 PRJNA528662 SRS4529461 SAMN11239136 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684268 GSM3684268 GEO Accession GSM3684268 SRX5566267 GSM3684269 SRP189280 PRJNA528662 SRS4529462 SAMN11239135 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684269 GSM3684269 GEO Accession GSM3684269 SRX5566268 GSM3684270 SRP189280 PRJNA528662 SRS4529463 SAMN11239134 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684270 GSM3684270 GEO Accession GSM3684270 SRX5566269 GSM3684271 SRP189280 PRJNA528662 SRS4529464 SAMN11239180 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684271 GSM3684271 GEO Accession GSM3684271 SRX5566270 GSM3684272 SRP189280 PRJNA528662 SRS4529465 SAMN11239179 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684272 GSM3684272 GEO Accession GSM3684272 SRX5566271 GSM3684273 SRP189280 PRJNA528662 SRS4529466 SAMN11239178 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684273 GSM3684273 GEO Accession GSM3684273 SRX5566272 GSM3684274 SRP189280 PRJNA528662 SRS4529467 SAMN11239177 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684274 GSM3684274 GEO Accession GSM3684274 SRX5566273 GSM3684275 SRP189280 PRJNA528662 SRS4529468 SAMN11239176 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684275 GSM3684275 GEO Accession GSM3684275 SRX5566274 GSM3684276 SRP189280 PRJNA528662 SRS4529469 SAMN11239175 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684276 GSM3684276 GEO Accession GSM3684276 SRX5566275 GSM3684277 SRP189280 PRJNA528662 SRS4529470 SAMN11239174 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684277 GSM3684277 GEO Accession GSM3684277 SRX5566276 GSM3684278 SRP189280 PRJNA528662 SRS4529471 SAMN11239173 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684278 GSM3684278 GEO Accession GSM3684278 SRX5566277 GSM3684279 SRP189280 PRJNA528662 SRS4529472 SAMN11239172 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684279 GSM3684279 GEO Accession GSM3684279 SRX5566278 GSM3684280 SRP189280 PRJNA528662 SRS4529473 SAMN11239171 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684280 GSM3684280 GEO Accession GSM3684280 SRX5566279 GSM3684281 SRP189280 PRJNA528662 SRS4529474 SAMN11239170 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684281 GSM3684281 GEO Accession GSM3684281 SRX5566280 GSM3684282 SRP189280 PRJNA528662 SRS4529475 SAMN11239169 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684282 GSM3684282 GEO Accession GSM3684282 SRX5566281 GSM3684283 SRP189280 PRJNA528662 SRS4529476 SAMN11239168 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684283 GSM3684283 GEO Accession GSM3684283 SRX5566282 GSM3684284 SRP189280 PRJNA528662 SRS4529477 SAMN11239167 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684284 GSM3684284 GEO Accession GSM3684284 SRX5566283 GSM3684285 SRP189280 PRJNA528662 SRS4529478 SAMN11239166 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684285 GSM3684285 GEO Accession GSM3684285 SRX5566284 GSM3684286 SRP189280 PRJNA528662 SRS4529479 SAMN11239165 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684286 GSM3684286 GEO Accession GSM3684286 SRX5566285 GSM3684287 SRP189280 PRJNA528662 SRS4529480 SAMN11239164 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684287 GSM3684287 GEO Accession GSM3684287 SRX5566286 GSM3684288 SRP189280 PRJNA528662 SRS4529481 SAMN11239163 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684288 GSM3684288 GEO Accession GSM3684288 SRX5566287 GSM3684289 SRP189280 PRJNA528662 SRS4529482 SAMN11239162 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684289 GSM3684289 GEO Accession GSM3684289 SRX5566288 GSM3684290 SRP189280 PRJNA528662 SRS4529483 SAMN11239161 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684290 GSM3684290 GEO Accession GSM3684290 SRX5566289 GSM3684291 SRP189280 PRJNA528662 SRS4529484 SAMN11239160 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684291 GSM3684291 GEO Accession GSM3684291 SRX5566290 GSM3684292 SRP189280 PRJNA528662 SRS4529485 SAMN11239202 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684292 GSM3684292 GEO Accession GSM3684292 SRX5566291 GSM3684293 SRP189280 PRJNA528662 SRS4529486 SAMN11239201 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684293 GSM3684293 GEO Accession GSM3684293 SRX5566292 GSM3684294 SRP189280 PRJNA528662 SRS4529487 SAMN11239159 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684294 GSM3684294 GEO Accession GSM3684294 SRX5566293 GSM3684295 SRP189280 PRJNA528662 SRS4529488 SAMN11239158 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684295 GSM3684295 GEO Accession GSM3684295 SRX5566294 GSM3684296 SRP189280 PRJNA528662 SRS4529489 SAMN11239157 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684296 GSM3684296 GEO Accession GSM3684296 SRX5566295 GSM3684297 SRP189280 PRJNA528662 SRS4529490 SAMN11239156 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684297 GSM3684297 GEO Accession GSM3684297 SRX5566296 GSM3684298 SRP189280 PRJNA528662 SRS4529491 SAMN11239203 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684298 GSM3684298 GEO Accession GSM3684298 SRX5566297 GSM3684299 SRP189280 PRJNA528662 SRS4529492 SAMN11239200 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684299 GSM3684299 GEO Accession GSM3684299 SRX5566298 GSM3684300 SRP189280 PRJNA528662 SRS4529493 SAMN11239199 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684300 GSM3684300 GEO Accession GSM3684300 SRX5566299 GSM3684301 SRP189280 PRJNA528662 SRS4529494 SAMN11239198 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684301 GSM3684301 GEO Accession GSM3684301 SRX5566300 GSM3684302 SRP189280 PRJNA528662 SRS4529495 SAMN11239197 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684302 GSM3684302 GEO Accession GSM3684302 SRX5566301 GSM3684303 SRP189280 PRJNA528662 SRS4529496 SAMN11239196 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684303 GSM3684303 GEO Accession GSM3684303 SRX5566302 GSM3684304 SRP189280 PRJNA528662 SRS4529497 SAMN11239195 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684304 GSM3684304 GEO Accession GSM3684304 SRX5566303 GSM3684305 SRP189280 PRJNA528662 SRS4529498 SAMN11239194 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684305 GSM3684305 GEO Accession GSM3684305 SRX5566304 GSM3684306 SRP189280 PRJNA528662 SRS4529499 SAMN11239193 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684306 GSM3684306 GEO Accession GSM3684306 SRX5566305 GSM3684307 SRP189280 PRJNA528662 SRS4529500 SAMN11239192 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684307 GSM3684307 GEO Accession GSM3684307 SRX5566306 GSM3684308 SRP189280 PRJNA528662 SRS4529501 SAMN11239191 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684308 GSM3684308 GEO Accession GSM3684308 SRX5566307 GSM3684309 SRP189280 PRJNA528662 SRS4529502 SAMN11239190 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684309 GSM3684309 GEO Accession GSM3684309 SRX5566308 GSM3684310 SRP189280 PRJNA528662 SRS4529503 SAMN11239189 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684310 GSM3684310 GEO Accession GSM3684310 SRX5566309 GSM3684311 SRP189280 PRJNA528662 SRS4529504 SAMN11239188 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 2500 gds 303684311 GSM3684311 GEO Accession GSM3684311 SRX5566310 GSM3684312 SRP189280 PRJNA528662 SRS4529505 SAMN11239187 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684312 GSM3684312 GEO Accession GSM3684312 SRX5566311 GSM3684313 SRP189280 PRJNA528662 SRS4529506 SAMN11239186 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684313 GSM3684313 GEO Accession GSM3684313 SRX5566312 GSM3684314 SRP189280 PRJNA528662 SRS4529507 SAMN11239185 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684314 GSM3684314 GEO Accession GSM3684314 SRX5566313 GSM3684315 SRP189280 PRJNA528662 SRS4529508 SAMN11239184 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684315 GSM3684315 GEO Accession GSM3684315 SRX5566314 GSM3684316 SRP189280 PRJNA528662 SRS4529509 SAMN11239183 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684316 GSM3684316 GEO Accession GSM3684316 SRX5566315 GSM3684317 SRP189280 PRJNA528662 SRS4529510 SAMN11239182 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684317 GSM3684317 GEO Accession GSM3684317 SRX5566316 GSM3684318 SRP189280 PRJNA528662 SRS4529511 SAMN11239181 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684318 GSM3684318 GEO Accession GSM3684318 SRX5566317 GSM3684319 SRP189280 PRJNA528662 SRS4529512 SAMN11239207 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684319 GSM3684319 GEO Accession GSM3684319 SRX5566318 GSM3684320 SRP189280 PRJNA528662 SRS4529513 SAMN11239206 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684320 GSM3684320 GEO Accession GSM3684320 SRX5566319 GSM3684321 SRP189280 PRJNA528662 SRS4529514 SAMN11239205 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684321 GSM3684321 GEO Accession GSM3684321 SRX5566320 GSM3684322 SRP189280 PRJNA528662 SRS4529515 SAMN11239204 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684322 GSM3684322 GEO Accession GSM3684322 SRX5566321 GSM3684323 SRP189280 PRJNA528662 SRS4529516 SAMN11239155 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684323 GSM3684323 GEO Accession GSM3684323 SRX5566322 GSM3684324 SRP189280 PRJNA528662 SRS4529517 SAMN11239154 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684324 GSM3684324 GEO Accession GSM3684324 SRX5566323 GSM3684325 SRP189280 PRJNA528662 SRS4529518 SAMN11239153 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684325 GSM3684325 GEO Accession GSM3684325 SRX5566324 GSM3684326 SRP189280 PRJNA528662 SRS4529519 SAMN11239152 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684326 GSM3684326 GEO Accession GSM3684326 SRX5566325 GSM3684327 SRP189280 PRJNA528662 SRS4529520 SAMN11239151 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684327 GSM3684327 GEO Accession GSM3684327 SRX5566326 GSM3684328 SRP189280 PRJNA528662 SRS4529521 SAMN11239150 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684328 GSM3684328 GEO Accession GSM3684328 SRX5566327 GSM3684329 SRP189280 PRJNA528662 SRS4529522 SAMN11239149 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684329 GSM3684329 GEO Accession GSM3684329 SRX5566328 GSM3684330 SRP189280 PRJNA528662 SRS4529523 SAMN11239148 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684330 GSM3684330 GEO Accession GSM3684330 SRX5566329 GSM3684331 SRP189280 PRJNA528662 SRS4529524 SAMN11239147 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684331 GSM3684331 GEO Accession GSM3684331 SRX5566330 GSM3684332 SRP189280 PRJNA528662 SRS4529525 SAMN11239146 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684332 GSM3684332 GEO Accession GSM3684332 SRX5566331 GSM3684333 SRP189280 PRJNA528662 SRS4529526 SAMN11239145 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684333 GSM3684333 GEO Accession GSM3684333 SRX5566332 GSM3684334 SRP189280 PRJNA528662 SRS4529527 SAMN11239144 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684334 GSM3684334 GEO Accession GSM3684334 SRX5566333 GSM3684335 SRP189280 PRJNA528662 SRS4529528 SAMN11239143 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684335 GSM3684335 GEO Accession GSM3684335 SRX5566334 GSM3684336 SRP189280 PRJNA528662 SRS4529529 SAMN11239142 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684336 GSM3684336 GEO Accession GSM3684336 SRX5566335 GSM3684337 SRP189280 PRJNA528662 SRS4529530 SAMN11239141 RNA-Seq TRANSCRIPTOMIC cDNA Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. Bone marrow cells were collected and RBC lysis was performed. Next, the cells were washed with FACS buffer and stained for analysis. For sorting of HSPCs the cells were enriched by magnetic depletion. Briefly, the BM cells were labeled with a cocktail of biotinylated antibodies (CD3, CD19, B220, CD11b, Gr1 and Ter119) and then with streptavidin-conjugated magnetic nanobeads (BioLegend). The Lineage- cells obtained were stained with following antibodies: anti-lineage (CD3, CD19, B220, CD11b, Gr1 and Ter119), anti-Sca1, anti-c-kit, anti-CD135, anti-CD48, anti-150, and then further purified by FACS sorting. The immunophenotypes of assayed hematopoietic cells were as follow: LSKs (Lin-Sca1+c-kit+), HSCs (CD135-CD48-CD150+LSK), MMP3 (CD135-CD48+CD150-LSK), MPP4 (CD135+ CD150-LSK), CLPs (Lin-Sca1loc-kitloIL-7Rα+CD135+), pro-B cells (B220+CD43+), pre-B cells (B220loCD43-), rec-B cells (B220hiCD43-), B cells (B220+CD19+), T cells (CD3+), myeloid cells (Mac1+Gr1+) and erythroid cells (Ter119+). For non-hematopoietic BM cell isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. The aRNA was reversely transcribed in order to generate libraries for sequencing, as previously described. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. Illumina HiSeq 3000 gds 303684337 GSM3684337 GEO Accession GSM3684337