GSM9384789: IgM_2 (RRBS); Mus musculus; Bisulfite-Seq
Spleen and dLN from influenza infected mice were homogenized, red blood cells lysed with ACK Lysis buffer (150mM NH4Cl, 10mM KHCO3 and 0.5M EDTA (pH 7.3)). Lymphocytes from influenza infected mice were stained for cell sorting. For each sample, DNA was harvested from 1,000-2,000 cells using Zymo Quick-DNA Microprep Kit (Zymo Research, D302;). The DNA was split at a 60:40 ratio and digested overnight with Mspl (New England BioLabs, R0106S) and Taql (New England BioLabs, R0149S) respectively to yield various sized sticky end fragments. The digested DNA fragments were combined, purified, and size selected using AMPure XP beads (Beckman Coulter, A63881). Lambda (λ) DNA (New England BioLabs, N3011S), CpG Methyltransferase New England BioLabs, M0226S) and ΦX174 RF I (PhiX) DNA (New England BioLabs, N3021S) were spiked in as bisulfite conversion internal controls. A-tailing, end repair, and methylated sequencing adaptor ligation was performed using the KAPA Hyper Prep Kit (KAPA Biosystems, KK850). Purified adaptor ligated genomic DNA was treated with sodium bisulfite to convert unmethylated cytosines to uracils with the EpiTech Bisulfite Kit (Qiagen, 5912). Bisulfite converted DNA was then PCR amplified (KAPA Biosystems, KK2802) with dual indexing Illumina compatible primers.