PRJDB5085 SAMD00057667 protonema1 OTHER TRANSCRIPTOMIC Oligo-dT poly(A) RNA was purified from 22 ~ 25 ug of total RNA with the FastTrack MAG mRNA micro purification kit (Life Technologies). After ethanol precipitation, 11 uL (220 ~ 250 ug) of poly(A) RNA and 1 uL of 50 uM T7-adA-dTCx3 (AATTGTAATACGACTCACTATAGGGAGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) primer were mixed, incubated at 74 C for 10 min, and then moved to ice. First-strand cDNAs were synthesized from 12 uL poly(A) RNA-primer mixture with 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 1 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Second-strand cDNAs were synthesized from 20 uL of first-strand cDNAs with 15 uL of Ligase buffer, 3 uL of 10 mM dNTPs, 1 uL of T4 DNA Ligase, 4 uL of E. coli polymerase I, 1 uL RNase H (Life Technologies), and 106 uL H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. After phenol/chloroform and chloroform extractions, a half-volume of 7.5 M ammonium acetate was added, and ethanol precipitation was performed. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O. Using the cDNAs as templates, RNA was synthesized using MEGAscript-T7 kit (Ambion) with 8 uL of double-stranded cDNAs, 2 uL of 75 mM ATP, 2 uL of 75 mM CTP, 2 uL of 75 mM GTP, 2 uL of 75 mM UTP, 2 uL of 10x Reaction Buffer, and 2 uL of Enzyme Mix-T7 at 37 C overnight. Then, 30 uL of LiCl Precipitation Solution in the kit was added into the solution, which was then incubated at -30 C for 30 min, and RNA was precipitated by centrifugation at 17,360 g at 4 C for 15 min. First-strand cDNAs were synthesized from 10 uL of 1 ~ 2 ug amplified RNA with 1 uL of 100 uM B-random primer (CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC), 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 2 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Then, 1 uL of RNase H (Life Technologies) was added to the solution, and the solution was incubated at 37 C for 20 min to degrade RNAs, followed by at 95 C for 5 min. Obtained single-strand cDNAs were purified with QIAquick PCR Purification Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride to remove B-random primers according to the manufacturer's protocol. Sterilized H2O and 1 uL of 100 uM adA-dTCx3 primer (AGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) were added to purified single-strand cDNAs to 50 uL final volume. The solution was incubated at 72 C for 6 min, and put on ice. Double-stranded cDNAs were synthesized from 50 uL single-stranded cDNA and adA-dTCx3 primer with 3 uL of 10 mM dNTPs, 4 uL of E. coli polymerase I, 15 uL of Ligase buffer (Life Technologies), and 78 uL of H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. Double-stranded cDNAs were purified by performing phenol/chloroform and chloroform extractions, and ethanol precipitation. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O, and sequencing libraries were synthesized by PCR from 2 ~ 4 uL of purified double-stranded cDNAs with 0.8 uL of 25 mM MgSO4, 1 uL of 2 mM dNTPs, 0.1 uL of 100 uM BioTEG/B-random primer (BioTEG-CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC, Integrated DNA Technologies), 0.1 uL of 100 uM adA-dTCx3 primer, 2 uL of 10x KOD plus buffer, 0.4 uL of KOD plus (Toyobo), and sterile H2O to 20 uL final volume, and incubated at 94 C for 2 min, followed by 24 cycles of 94 C for 15 sec, 57 C for 30 sec, 68 C for 40 sec. Sequencing libraries were subject to electrophoresis in a 2% agarose gel, the region between 200 to 800 bp was excised, and cDNAs were purified with the MinElute Gel Extraction Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride as described above. Sequencing samples were prepared from sequencing libraries, and were subjected to massive parallel sequencing using a Genome Sequencer 20 (Roche) according to the manufacturer's protocol. 168 0 Application Read Forward 1 454 GS 20 PRJDB5085 SAMD00057668 Gametophores OTHER TRANSCRIPTOMIC Oligo-dT poly(A) RNA was purified from 22 ~ 25 ug of total RNA with the FastTrack MAG mRNA micro purification kit (Life Technologies). After ethanol precipitation, 11 uL (220 ~ 250 ug) of poly(A) RNA and 1 uL of 50 uM T7-adA-dTCx3 (AATTGTAATACGACTCACTATAGGGAGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) primer were mixed, incubated at 74 C for 10 min, and then moved to ice. First-strand cDNAs were synthesized from 12 uL poly(A) RNA-primer mixture with 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 1 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Second-strand cDNAs were synthesized from 20 uL of first-strand cDNAs with 15 uL of Ligase buffer, 3 uL of 10 mM dNTPs, 1 uL of T4 DNA Ligase, 4 uL of E. coli polymerase I, 1 uL RNase H (Life Technologies), and 106 uL H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. After phenol/chloroform and chloroform extractions, a half-volume of 7.5 M ammonium acetate was added, and ethanol precipitation was performed. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O. Using the cDNAs as templates, RNA was synthesized using MEGAscript-T7 kit (Ambion) with 8 uL of double-stranded cDNAs, 2 uL of 75 mM ATP, 2 uL of 75 mM CTP, 2 uL of 75 mM GTP, 2 uL of 75 mM UTP, 2 uL of 10x Reaction Buffer, and 2 uL of Enzyme Mix-T7 at 37 C overnight. Then, 30 uL of LiCl Precipitation Solution in the kit was added into the solution, which was then incubated at -30 C for 30 min, and RNA was precipitated by centrifugation at 17,360 g at 4 C for 15 min. First-strand cDNAs were synthesized from 10 uL of 1 ~ 2 ug amplified RNA with 1 uL of 100 uM B-random primer (CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC), 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 2 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Then, 1 uL of RNase H (Life Technologies) was added to the solution, and the solution was incubated at 37 C for 20 min to degrade RNAs, followed by at 95 C for 5 min. Obtained single-strand cDNAs were purified with QIAquick PCR Purification Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride to remove B-random primers according to the manufacturer's protocol. Sterilized H2O and 1 uL of 100 uM adA-dTCx3 primer (AGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) were added to purified single-strand cDNAs to 50 uL final volume. The solution was incubated at 72 C for 6 min, and put on ice. Double-stranded cDNAs were synthesized from 50 uL single-stranded cDNA and adA-dTCx3 primer with 3 uL of 10 mM dNTPs, 4 uL of E. coli polymerase I, 15 uL of Ligase buffer (Life Technologies), and 78 uL of H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. Double-stranded cDNAs were purified by performing phenol/chloroform and chloroform extractions, and ethanol precipitation. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O, and sequencing libraries were synthesized by PCR from 2 ~ 4 uL of purified double-stranded cDNAs with 0.8 uL of 25 mM MgSO4, 1 uL of 2 mM dNTPs, 0.1 uL of 100 uM BioTEG/B-random primer (BioTEG-CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC, Integrated DNA Technologies), 0.1 uL of 100 uM adA-dTCx3 primer, 2 uL of 10x KOD plus buffer, 0.4 uL of KOD plus (Toyobo), and sterile H2O to 20 uL final volume, and incubated at 94 C for 2 min, followed by 24 cycles of 94 C for 15 sec, 57 C for 30 sec, 68 C for 40 sec. Sequencing libraries were subject to electrophoresis in a 2% agarose gel, the region between 200 to 800 bp was excised, and cDNAs were purified with the MinElute Gel Extraction Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride as described above. Sequencing samples were prepared from sequencing libraries, and were subjected to massive parallel sequencing using a Genome Sequencer 20 (Roche) according to the manufacturer's protocol. 168 0 Application Read Forward 1 454 GS 20 PRJDB5085 SAMD00057669 Reprogramming OTHER TRANSCRIPTOMIC Oligo-dT poly(A) RNA was purified from 22 ~ 25 ug of total RNA with the FastTrack MAG mRNA micro purification kit (Life Technologies). After ethanol precipitation, 11 uL (220 ~ 250 ug) of poly(A) RNA and 1 uL of 50 uM T7-adA-dTCx3 (AATTGTAATACGACTCACTATAGGGAGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) primer were mixed, incubated at 74 C for 10 min, and then moved to ice. First-strand cDNAs were synthesized from 12 uL poly(A) RNA-primer mixture with 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 1 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Second-strand cDNAs were synthesized from 20 uL of first-strand cDNAs with 15 uL of Ligase buffer, 3 uL of 10 mM dNTPs, 1 uL of T4 DNA Ligase, 4 uL of E. coli polymerase I, 1 uL RNase H (Life Technologies), and 106 uL H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. After phenol/chloroform and chloroform extractions, a half-volume of 7.5 M ammonium acetate was added, and ethanol precipitation was performed. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O. Using the cDNAs as templates, RNA was synthesized using MEGAscript-T7 kit (Ambion) with 8 uL of double-stranded cDNAs, 2 uL of 75 mM ATP, 2 uL of 75 mM CTP, 2 uL of 75 mM GTP, 2 uL of 75 mM UTP, 2 uL of 10x Reaction Buffer, and 2 uL of Enzyme Mix-T7 at 37 C overnight. Then, 30 uL of LiCl Precipitation Solution in the kit was added into the solution, which was then incubated at -30 C for 30 min, and RNA was precipitated by centrifugation at 17,360 g at 4 C for 15 min. First-strand cDNAs were synthesized from 10 uL of 1 ~ 2 ug amplified RNA with 1 uL of 100 uM B-random primer (CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC), 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 2 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Then, 1 uL of RNase H (Life Technologies) was added to the solution, and the solution was incubated at 37 C for 20 min to degrade RNAs, followed by at 95 C for 5 min. Obtained single-strand cDNAs were purified with QIAquick PCR Purification Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride to remove B-random primers according to the manufacturer's protocol. Sterilized H2O and 1 uL of 100 uM adA-dTCx3 primer (AGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) were added to purified single-strand cDNAs to 50 uL final volume. The solution was incubated at 72 C for 6 min, and put on ice. Double-stranded cDNAs were synthesized from 50 uL single-stranded cDNA and adA-dTCx3 primer with 3 uL of 10 mM dNTPs, 4 uL of E. coli polymerase I, 15 uL of Ligase buffer (Life Technologies), and 78 uL of H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. Double-stranded cDNAs were purified by performing phenol/chloroform and chloroform extractions, and ethanol precipitation. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O, and sequencing libraries were synthesized by PCR from 2 ~ 4 uL of purified double-stranded cDNAs with 0.8 uL of 25 mM MgSO4, 1 uL of 2 mM dNTPs, 0.1 uL of 100 uM BioTEG/B-random primer (BioTEG-CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC, Integrated DNA Technologies), 0.1 uL of 100 uM adA-dTCx3 primer, 2 uL of 10x KOD plus buffer, 0.4 uL of KOD plus (Toyobo), and sterile H2O to 20 uL final volume, and incubated at 94 C for 2 min, followed by 24 cycles of 94 C for 15 sec, 57 C for 30 sec, 68 C for 40 sec. Sequencing libraries were subject to electrophoresis in a 2% agarose gel, the region between 200 to 800 bp was excised, and cDNAs were purified with the MinElute Gel Extraction Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride as described above. Sequencing samples were prepared from sequencing libraries, and were subjected to massive parallel sequencing using a Genome Sequencer 20 (Roche) according to the manufacturer's protocol. 168 0 Application Read Forward 1 454 GS 20 PRJDB5085 SAMD00057670 Gametangia OTHER TRANSCRIPTOMIC Oligo-dT poly(A) RNA was purified from 22 ~ 25 ug of total RNA with the FastTrack MAG mRNA micro purification kit (Life Technologies). After ethanol precipitation, 11 uL (220 ~ 250 ug) of poly(A) RNA and 1 uL of 50 uM T7-adA-dTCx3 (AATTGTAATACGACTCACTATAGGGAGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) primer were mixed, incubated at 74 C for 10 min, and then moved to ice. First-strand cDNAs were synthesized from 12 uL poly(A) RNA-primer mixture with 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 1 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Second-strand cDNAs were synthesized from 20 uL of first-strand cDNAs with 15 uL of Ligase buffer, 3 uL of 10 mM dNTPs, 1 uL of T4 DNA Ligase, 4 uL of E. coli polymerase I, 1 uL RNase H (Life Technologies), and 106 uL H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. After phenol/chloroform and chloroform extractions, a half-volume of 7.5 M ammonium acetate was added, and ethanol precipitation was performed. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O. Using the cDNAs as templates, RNA was synthesized using MEGAscript-T7 kit (Ambion) with 8 uL of double-stranded cDNAs, 2 uL of 75 mM ATP, 2 uL of 75 mM CTP, 2 uL of 75 mM GTP, 2 uL of 75 mM UTP, 2 uL of 10x Reaction Buffer, and 2 uL of Enzyme Mix-T7 at 37 C overnight. Then, 30 uL of LiCl Precipitation Solution in the kit was added into the solution, which was then incubated at -30 C for 30 min, and RNA was precipitated by centrifugation at 17,360 g at 4 C for 15 min. First-strand cDNAs were synthesized from 10 uL of 1 ~ 2 ug amplified RNA with 1 uL of 100 uM B-random primer (CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC), 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 2 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Then, 1 uL of RNase H (Life Technologies) was added to the solution, and the solution was incubated at 37 C for 20 min to degrade RNAs, followed by at 95 C for 5 min. Obtained single-strand cDNAs were purified with QIAquick PCR Purification Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride to remove B-random primers according to the manufacturer's protocol. Sterilized H2O and 1 uL of 100 uM adA-dTCx3 primer (AGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) were added to purified single-strand cDNAs to 50 uL final volume. The solution was incubated at 72 C for 6 min, and put on ice. Double-stranded cDNAs were synthesized from 50 uL single-stranded cDNA and adA-dTCx3 primer with 3 uL of 10 mM dNTPs, 4 uL of E. coli polymerase I, 15 uL of Ligase buffer (Life Technologies), and 78 uL of H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. Double-stranded cDNAs were purified by performing phenol/chloroform and chloroform extractions, and ethanol precipitation. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O, and sequencing libraries were synthesized by PCR from 2 ~ 4 uL of purified double-stranded cDNAs with 0.8 uL of 25 mM MgSO4, 1 uL of 2 mM dNTPs, 0.1 uL of 100 uM BioTEG/B-random primer (BioTEG-CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC, Integrated DNA Technologies), 0.1 uL of 100 uM adA-dTCx3 primer, 2 uL of 10x KOD plus buffer, 0.4 uL of KOD plus (Toyobo), and sterile H2O to 20 uL final volume, and incubated at 94 C for 2 min, followed by 24 cycles of 94 C for 15 sec, 57 C for 30 sec, 68 C for 40 sec. Sequencing libraries were subject to electrophoresis in a 2% agarose gel, the region between 200 to 800 bp was excised, and cDNAs were purified with the MinElute Gel Extraction Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride as described above. Sequencing samples were prepared from sequencing libraries, and were subjected to massive parallel sequencing using a Genome Sequencer 20 (Roche) according to the manufacturer's protocol. 168 0 Application Read Forward 1 454 GS 20 PRJDB5085 SAMD00057671 Sporophytes OTHER TRANSCRIPTOMIC Oligo-dT poly(A) RNA was purified from 22 ~ 25 ug of total RNA with the FastTrack MAG mRNA micro purification kit (Life Technologies). After ethanol precipitation, 11 uL (220 ~ 250 ug) of poly(A) RNA and 1 uL of 50 uM T7-adA-dTCx3 (AATTGTAATACGACTCACTATAGGGAGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) primer were mixed, incubated at 74 C for 10 min, and then moved to ice. First-strand cDNAs were synthesized from 12 uL poly(A) RNA-primer mixture with 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 1 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Second-strand cDNAs were synthesized from 20 uL of first-strand cDNAs with 15 uL of Ligase buffer, 3 uL of 10 mM dNTPs, 1 uL of T4 DNA Ligase, 4 uL of E. coli polymerase I, 1 uL RNase H (Life Technologies), and 106 uL H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. After phenol/chloroform and chloroform extractions, a half-volume of 7.5 M ammonium acetate was added, and ethanol precipitation was performed. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O. Using the cDNAs as templates, RNA was synthesized using MEGAscript-T7 kit (Ambion) with 8 uL of double-stranded cDNAs, 2 uL of 75 mM ATP, 2 uL of 75 mM CTP, 2 uL of 75 mM GTP, 2 uL of 75 mM UTP, 2 uL of 10x Reaction Buffer, and 2 uL of Enzyme Mix-T7 at 37 C overnight. Then, 30 uL of LiCl Precipitation Solution in the kit was added into the solution, which was then incubated at -30 C for 30 min, and RNA was precipitated by centrifugation at 17,360 g at 4 C for 15 min. First-strand cDNAs were synthesized from 10 uL of 1 ~ 2 ug amplified RNA with 1 uL of 100 uM B-random primer (CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC), 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 2 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Then, 1 uL of RNase H (Life Technologies) was added to the solution, and the solution was incubated at 37 C for 20 min to degrade RNAs, followed by at 95 C for 5 min. Obtained single-strand cDNAs were purified with QIAquick PCR Purification Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride to remove B-random primers according to the manufacturer's protocol. Sterilized H2O and 1 uL of 100 uM adA-dTCx3 primer (AGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) were added to purified single-strand cDNAs to 50 uL final volume. The solution was incubated at 72 C for 6 min, and put on ice. Double-stranded cDNAs were synthesized from 50 uL single-stranded cDNA and adA-dTCx3 primer with 3 uL of 10 mM dNTPs, 4 uL of E. coli polymerase I, 15 uL of Ligase buffer (Life Technologies), and 78 uL of H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. Double-stranded cDNAs were purified by performing phenol/chloroform and chloroform extractions, and ethanol precipitation. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O, and sequencing libraries were synthesized by PCR from 2 ~ 4 uL of purified double-stranded cDNAs with 0.8 uL of 25 mM MgSO4, 1 uL of 2 mM dNTPs, 0.1 uL of 100 uM BioTEG/B-random primer (BioTEG-CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC, Integrated DNA Technologies), 0.1 uL of 100 uM adA-dTCx3 primer, 2 uL of 10x KOD plus buffer, 0.4 uL of KOD plus (Toyobo), and sterile H2O to 20 uL final volume, and incubated at 94 C for 2 min, followed by 24 cycles of 94 C for 15 sec, 57 C for 30 sec, 68 C for 40 sec. Sequencing libraries were subject to electrophoresis in a 2% agarose gel, the region between 200 to 800 bp was excised, and cDNAs were purified with the MinElute Gel Extraction Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride as described above. Sequencing samples were prepared from sequencing libraries, and were subjected to massive parallel sequencing using a Genome Sequencer 20 (Roche) according to the manufacturer's protocol. 168 0 Application Read Forward 1 454 GS 20 PRJDB5085 SAMD00057667 protonema2 OTHER TRANSCRIPTOMIC Oligo-dT poly(A) RNA was purified from 22 ~ 25 ug of total RNA with the FastTrack MAG mRNA micro purification kit (Life Technologies). After ethanol precipitation, 11 uL (220 ~ 250 ug) of poly(A) RNA and 1 uL of 50 uM T7-adA-dTCx3 (AATTGTAATACGACTCACTATAGGGAGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) primer were mixed, incubated at 74 C for 10 min, and then moved to ice. First-strand cDNAs were synthesized from 12 uL poly(A) RNA-primer mixture with 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 1 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Second-strand cDNAs were synthesized from 20 uL of first-strand cDNAs with 15 uL of Ligase buffer, 3 uL of 10 mM dNTPs, 1 uL of T4 DNA Ligase, 4 uL of E. coli polymerase I, 1 uL RNase H (Life Technologies), and 106 uL H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. After phenol/chloroform and chloroform extractions, a half-volume of 7.5 M ammonium acetate was added, and ethanol precipitation was performed. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O. Using the cDNAs as templates, RNA was synthesized using MEGAscript-T7 kit (Ambion) with 8 uL of double-stranded cDNAs, 2 uL of 75 mM ATP, 2 uL of 75 mM CTP, 2 uL of 75 mM GTP, 2 uL of 75 mM UTP, 2 uL of 10x Reaction Buffer, and 2 uL of Enzyme Mix-T7 at 37 C overnight. Then, 30 uL of LiCl Precipitation Solution in the kit was added into the solution, which was then incubated at -30 C for 30 min, and RNA was precipitated by centrifugation at 17,360 g at 4 C for 15 min. First-strand cDNAs were synthesized from 10 uL of 1 ~ 2 ug amplified RNA with 1 uL of 100 uM B-random primer (CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC), 4 uL of First-Strand Buffer, 2 uL of 100 mM DTT, 2 uL of SuperScript II (Life Technologies), and 1 uL of 10 mM dNTPs at 42 C for 1 hr. Then, 1 uL of RNase H (Life Technologies) was added to the solution, and the solution was incubated at 37 C for 20 min to degrade RNAs, followed by at 95 C for 5 min. Obtained single-strand cDNAs were purified with QIAquick PCR Purification Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride to remove B-random primers according to the manufacturer's protocol. Sterilized H2O and 1 uL of 100 uM adA-dTCx3 primer (AGACCATCTCATCCCTGCGTGTCCCATCTGTTCCCTCCCTGTCTCAGTTTTTTCTTTTTTCTTTTTTCTTTTVN) were added to purified single-strand cDNAs to 50 uL final volume. The solution was incubated at 72 C for 6 min, and put on ice. Double-stranded cDNAs were synthesized from 50 uL single-stranded cDNA and adA-dTCx3 primer with 3 uL of 10 mM dNTPs, 4 uL of E. coli polymerase I, 15 uL of Ligase buffer (Life Technologies), and 78 uL of H2O at 16 C for 2 hr. Then, 2 uL of T4 DNA polymerase (Takara) was added to the solution and incubated at 16 C for 5 min. The synthesis was terminated with 10 uL of 0.5 M EDTA. Double-stranded cDNAs were purified by performing phenol/chloroform and chloroform extractions, and ethanol precipitation. Precipitated double-stranded cDNAs were dissolved in 8 uL H2O, and sequencing libraries were synthesized by PCR from 2 ~ 4 uL of purified double-stranded cDNAs with 0.8 uL of 25 mM MgSO4, 1 uL of 2 mM dNTPs, 0.1 uL of 100 uM biotin labeled B-random primer (BioTEG-CCTATCCCCTGTGTGCCTTGCCTATCCCCTGTTGCGTGTCTCAGNNNNNNC, Operon), 0.1 uL of 100 uM adA-dTCx3 primer, 2 uL of 10x KOD plus buffer, 0.4 uL of KOD plus (Toyobo), and sterile H2O to 20 uL final volume, and incubated at 94 C for 2 min, followed by 24 cycles of 94 C for 15 sec, 57 C for 30 sec, 68 C for 40 sec. Sequencing libraries were subject to electrophoresis in a 2% agarose gel, the region between 200 to 800 bp was excised, and cDNAs were purified with the MinElute Gel Extraction Kit (Qiagen). During the purification, cDNA-bound columns were washed with 750 uL of 35% guanidine hydrochloride as described above. Sequencing samples were prepared from sequencing libraries, and were subjected to massive parallel sequencing using a Genome Sequencer 20 (Roche) according to the manufacturer's protocol. 168 0 Application Read Forward 1 454 GS 20