title: GSM4151108: Bulk HBS 32-cell embryos Library 1; Mus musculus; OTHER
library_construction_protocol: Embryos were picked manually and deposited into a 0.5 mL eppendorf with lysis buffer and Vapor-lock. Hairpin bisulfite sequencing was performed on bulk mouse embryos samples ( 2-cell to 64 cell stage). The bulk embryos were treated with protease treatment (1 uL of 25 ug/uL Qiagen Protease, 1 uL of 10X NEB Buffer 4, and 8 uL of nuclease free water). Then, 0.5 ng of genomic DNA was digested with 20 mL of MspI master mix (1 uL of MspI R0104L, 2 mL 10X NEB Cutsmart buffer in a total volume of 20 uL) and incubated at 37 C for an hour. After digestion, the digested genomic DNA was ligated with 1 uL of 10 uM phosphorylated hairpin oligo and ligation master mix (1 mL of NEB T4 ligase M0202M, 1 uL of 10X NEB T4 Ligase buffer, 2 uL of 10mM ATP, 5 uL of nuclease free water) and incubated overnight at 16 C. For purification of the ligation mixture, we used Dynabeads™ M-280 Streptavidin and followed the recommended protocol with the following exceptions: the bead-ligation mixture was incubated for at least an hour at RT on a rotator and cold 10 mM Tris-HCl wash step was included. Subsequently, we performed BS-seq on the sample using the protocol Clark, et al. 2017. After sequencing the libraries on a Miseq 300x300 or NextSeq 75x75 pair-end kit, we used HBS-tools (Sun, MA et al. 2015) and custom perl scripts were used to analyze the methylated CpG dyads. Hairpin oligo (G/iMe-dC/iMe-dC/G/iMe-dC/iMe-dC/GG/iMe-dC/GG/iMe-dC/AAG/iBiodT/GAAG/iMe-dC/iMe-dC/G/iMe-dC/iMe-dC/GG/iMe-dC/G) was resuspended in 100 uM of Low-TE. The hairpin oligo was phosphorylated (1 uL of 100 uM hairpin oligo, 3 uL of 10X T4 Ligase Buffer, 1 uL T4 PNK and 5 uL of nuclease free water) and incubated at 37 C for an hour. Subsequently the phosphorylated oligo heated at 94 C and placed in ice water to generate the loop.
attributes.tag: GEO Accession
attributes.value: GSM4151108