GSM9890322: CasTuner XO sgXist ctrl Day4 Rep2; Mus musculus; Bisulfite-Seq
Genomic DNA was extracted from harvested cells using the DNeasy Blood & Tissue kit (Qiagen). 1μg of DNA were bisulfite-converted with EZ DNA Methylation-Gold kit (Zymo). One fifth of the eluted DNA was used per amplicon PCR. Primers for bisulfite-converted DNA targeting the Xist-promoter proximal CpG island (chrX:103,482,090-103,482,350 mm10 from UCSC Genome Browser) were designed with MethPrimer2.0 (Li & Dahiya, 2002). PCR was performed with EpiTaq polymerase (Takara), using 25mM MgCl2. DNA was amplified for 27 cycles, at an annealing temperature of 53oC. PCR reactions of each sample were pooled (total volume for Xist amplicons) and cleaned with MinElute Reaction Cleanup kit (Qiagen) (2 columns per sample). Libraries were prepared with NEBNext Ultra II DNA Library Prep kit (New England Biolabs), indexed with NEBNext Multiplex Oligos for Illumina Dual Index Set 1 (New England Biolabs) and cleaned with AMPure XP beads (Beckman Coulter). Concentration and size distribution of libraries were analyzed with Qubit 1x dsDNA HS (Thermo Fisher Scientific) and HS DNA D1000 Tapestation (Agilent), respectively. Libraries were sequenced 2x150bp paired-end with MiSeq, DNBSEQ G400 or Aviti sequencers.