GSM8508184: WGBS3_3ppm_TP57-5_M_KO; Mus musculus; Bisulfite-Seq
Genomic DNA (gDNA) was extracted from embryonic brains using the Purelink Genomic DNA mini kit (Invitrogen, K182001) from individual E11.5 brains . DNA quality was assessed with NanoDdrop One (Thermo Fisher) , and absence of RNA contamination was confirmed with a 0.8% agarose gel stained with SYBR Safe. 400 ng of genomic DNA was sonicated on a Covaris LE220 with settings targeting an average size of 400 bp. Prior to bisulfite conversion, 0.5% unmethylated Lambda gDNA was spiked in and DNA quantification performed using Picogreen. 400 ng of sheared DNA was bisulfite converted with the Zymo EZ DNA Methylation Gold kit (cat# D5005, VWR) following the product manual. Estimating a 75% recovery rate, 50 ng of deaminated DNA was used for library prep with the IDT xGen™ Methyl-Seq DNA library prep kit (cat#10009824). Briefly, gDNA was heat-denatured and the single stranded DNA was used as template for proprietary adaptase stub tailing and adapter ligation prior to full-length adapter addition during the indexing PCR amplification of the library. Following product purification, the resulting libraries were quantitated by Picogreen and fragment size assessed with the Agilent 2100 Bioanalyzer. All samples were pooled equimolarly and re-quantitated by qPCR using the Applied Biosystems ViiA7 Quantitative PCR instrument and a KAPA Library Quant Kit (p/n KK4824). Using the concentration from the ViiA7™ qPCR machine above, 150 pM of equimolarly pooled library was loaded onto one lane of the NovaSeq S4 flowcell (Illumina p/n 20028312) following the XP Workflow protocol (Illumina kit p/n 20043131) and amplified by exclusion amplification onto a nanowell-designed, patterned flowcell using the Illumina NovaSeq 6000 sequencing instrument. PhiX Control v3 adapter-ligated library (Illumina p/n FC-110-3001) was spiked-in at 2% by weight to ensure balanced diversity and to monitor clustering and sequencing performance. A paired-end 150 bp cycle run was used to sequence an average of 400 million read pairs (~40X coverage) per sample.