GSM6935499: WGBS STB_WGBS_SCNT_rep1; Macaca fascicularis; Bisulfite-Seq
For the placenta tissues, their whole genome was extracted with the TIANamp Genomic DNA Kit (DP304-03) under the manufactures construction. For the STBThe oocytes were lysed in the lysis buffer and then whole genome were For the WGBS of placenta, 1 µg genomic DNA was fragmented to 200-300bp and purified using MiniElute PCR Purification Kit (QIAGEN). The resulted DNA was incubated at 20 ℃ with End Repair Mix and then purified. After end repair, the library was combined with A-Tailing Mix and incubated at 37 ℃ and then purified. Next, methylated adaptors were ligated to the Adenylate 3'Ends DNA and purified again. The above library was then undergoing bisulfite treatment and purified with Methylation-Gold kit (ZYMO). After that, fragments ranging from 320 to 420 bp were purified using agarose gel electrophoresis. The library was then amplified using PCR. Finally, the purified DNA products was qualified using the Agilent Technologies 2100 bioanalyzer and ABI StepOnePlus Real-Time PCR system. The library was sequenced in pair-end on the DNBseq platform.     STB cells were seeded into lysis buffer by mouth pipette and subjected to bisulfite conversion using the EZ DNA Methylation Direct Kit (Zymo Research, D5020). A small amount of unmethylated Lambda DNA (Promega, D152A) was added to each sample before bisulfite conversion to serve as spike-in controls for evaluating bisulfite conversion efficiency. After column-based purification, DNA was complemented with the random primer Preamp and Klenow polymerase (Enzymatics, P7010-HC-L). This random priming was repeated five times in total. Second strands were synthesized using another random primer, Adapter 2. Final libraries were generated after PCR amplification with the Illumina universal PCR primer and Illumina indexed primer, and were then purified using SPRI beads. Final libraries were subjected to paired-end 150 bp sequencing on a NovaSeq 6000 sequencer (Illumina) with PhiX spike-in control.     For the WGBS library construction of oocyte, a single oocyte was collected for each replication. A total of 2 GV oocytes and 3 MII oocytes were collected in this research. After cells were lysed using cell lysis buffer, bisulfite treatment was performed and the resultant DNA was purified using PureLink PCR Micro Kit (InvitrogenTM, K310050). The purified DNA was added with oligo1, followed by PCR. Then, the single strand DNA template was digested using polymerase, and the PCR products was purified using DynabeadsM-280 Streptavidin. The beads were resuspended in reaction mix with oligo2, the products were then purified again using Agencourt RNAClean XP beads. The purified DNA products were amplified using primers containing illumina specific primer and index. Finally, the amplified PCR products were further purified and eluted in elution buffer. The library was qualified using Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System (TaqMan Prob). The qualified DNA library sequenced using DNBseq platform.