WGBS in WT and Dnmt3a KO HSCs naive and after 1-month of infection with M. avium
Description
100000 LT-HSCs (LK CD150+ CD48- were sorted into lysis buffer from the pools of naive or 1-month (M. avium) infected WT/Dnmt3a-/- mice (n=7-8 per group). Around 300 ng of DNA per group was extracted with AllPrep DNA/RNA Mini Kit. NEBNext Ultra II DNA library prep kit was used for library preparation. Bisulfite conversion step was added after “methylated” adaptor ligation and USER excision. Then, methylated adaptor ligated DNA fragment was treated with bisulfite conversion using EZ DNA methylation-lightning kit. These modified DNA fragments were then amplified with index primers with Kapa HiFi urasil+ specialized polymerase for bisulfite converted DNA. For WGBS library sequencing, 150-high output kit from illumine was used. Samples (400 million reads/sample (15-20X coverage)) were run in NovaSeq S1 FC (2, lanes 1,600 Million reads). Quality control was conducted using FastQC and CGmapTools. Adapter and quality trimmed reads, processed using trim_galore, were aligned to GRCm38 reference genome using bs_seeker2 ,based on bowtie read aligner. The average alignment rate for all samples was 48%. Duplicate reads were removed using MarkDuplicates from the Picard tool set, resulting in an average of 11.5x coverage across the entire genome for all of the samples (minimum 10.7x maximum 12.5x for individual samples). Raw methylation calls were extracted from pre-processed reads using bs_seeker2. Differentially methylated regions (DMRs) were identified with CGmapTools using all CG sites with a minimum of 4x and maximum of 100x coverage and Benjamini & Hochberg correction for multiple testing. We assumed 0.01 significance level and retained only sites that showed at least 0.1 methylation level difference. Visualizations of DMRs were created with Gviz ,using GENCODE mouse gene annotation database v24