GSM2991557: alpha-Amanitin_8cell_embryo1_4; Homo sapiens; Bisulfite-Seq
The single cell of early embryos were picked into PCR tube containing lysis buffer (10 mM Tris-HCl pH 7.4, 0.25 mM EDTA, 0.25 mM PMSF, 0.5% NP-40, 10 mM DDT, 50 mM NaCl and 1 pg unmethylated lambda DNA) by mouth pipette, in order to release genomic DNA. After pick a single cell into lysis buffer, the tube was vortexed violently for 30 s to lyse efficiently. After spinning down, the lysate was incubated in 5 U GpC MTase (NEB, M0227L) and 160 μM S-adenosylmethionine at 37 ℃ for 45 min and 65 ℃ for 25 min. To release the genomic DNA, 0.5 μl 20 mg/ul protease (QIAGEN) was added and the sample was incubated at 50 ℃ for 3 hrs, 20 ℃ for 20 min. The scCOOL-seq libraries were constructed with slight modification. Briefly, the bisulfite conversion was performed using EZ-96 DNA Methylation-DirectTMMagPrep (Zymo, D5044) according to the user guide. After purification, 10 rounds of amplification were completed in the presence of Klenow exo- (Enzymics) and scBS-seq-P5-N6-oligo1  (CTACACGACGCTCTTCCGATCTNNNNNN). The amplification product was purified by 0.8x Agencourt AMPure XP beads (Beckman). The second strand was synthesied using scBS-seq-P7-N6-oligo2 (AGACGTGTGCTCTTCCGATCTNNNNN), followed by 0.8x Agencourt AMPure XP beads (Beckman). 13 cycles of PCR were performed to amplify the library using index primer and universal primer (NEB). The libraries were sequenced on Illumina Hiseq 4000 platform on pair-end 150 bp mode.
GEO Accession
GSM2991557