GSM4678823: DNA_Granulosa_sc57; Homo sapiens; Bisulfite-Seq
For the isolation of human growing oocytes and ovarian somatic cells, fresh ovarian tissue (~1cm3) was cut into pieces after the blood vessels and cysts were removed. Then, pieces of ovarian tissue were dissociated in 2ml of 1× DPBS (Gibco) with 5% collagenase (Gibco) at 37°C for 1 h, 1000× rpm on a thermo incubator. After the first round dissociation, the sample was filtrated through a 100-µm cell strainer (Biololgix Group). Single cells were centrifuged at 300× g for 5 min and resuspended in 2ml LFP medium (1× L15 medium (Gibco), 10% FBS (Vistec) and 1% penicillin–streptomycin (Gibco)) before picking up. Next, the undissociated tissue sample underwent the second round digestion by 2ml TrypLE™ Express (Gibco) at 37°C for 30 min, 1000× rpm on a thermo incubator. Then the digested sample was filtrated through a 100-µm cell strainer (Biololgix Group). Finally, single cells were centrifuged at 300× g for 5 min and resuspended in 2ml LFP medium before selection. Individual cell was picked and subjected to the scChaRM-seq steps. For mature human oocytes, zona pellucida was removed by Tyrode's Solution (Sigma). Then, oocytes were washed in 1× DPBS with 1% BSA by several times under a stereomicroscope. Individual oocyte was picked and subjected to the scChaRM-seq steps. For scChaRM-seq, single cells were individually picked into 0.25-ml PCR tubes containing 1.825 µl of ice-cold lysis buffer. Then, in vitro GpC methylation was performed by adding 2.5 U of M.CviPI (New England Biolabs) and 160 μM SAM (New England Biolabs)directly to the single-cell lysate. Samples were incubated at 37°C for 15 min, and the reaction was stopped by adding 5 μl of RLT buffer (Qiagen). Single-cell mRNA was captured by biotin-tagged and barcoded-oligo-dT primer conjugated to magnetic streptavidin beads (Invitrogen), and the gDNA supernatant was transferred into another PCR tube for methylome analysis. The mRNA separated from single cells was subjected to cDNA synthesis, PCR amplification and library construction according to our previously reported protocol. The corresponding gDNA from single cells was first purified by AMPureXP beads (Beckman Coulter), then subjected to bisulfite conversion (Zymo Research) and DNA library construction by our recently published separated (TAILS) method. For SCRB-seq, single cells were picked, lysed and subjected to first-strand cDNA synthesis as reported previously. Then, second-strand cDNA was synthesized, amplified and fragmented. An RNA-seq library was prepared by following the instruction manual of the NEBNext UltraII DNA Library Prep Kit for Illumina (New England Biolabs). Finally, the libraries were checked, pooled and sequenced with paired-end 150-bp reads on the Illumina HiSeq X-Ten platform (Novogene).
GEO Accession
GSM4678823