title: GSM2850584: RRBS_NP3; Mus musculus; Bisulfite-Seq
library_construction_protocol: 48 hours post-aspiration, all groups were euthanized for isolation of lung CD4+ T cells by magnetic bead pre-enrichment and fluorescence-activated cell sorting. RNA and genomic DNA were obtained using the AllPrep DNA/RNA Micro Kit (Qiagen). Genomic DNA was quantified with a Qubit 3.0 instrument. ~50-200 ng of genomic DNA was then digested with the restriction endonuclease MspI (New England BioLabs). Resulting fragments underwent size selection for fragments ~100-250-bp in length using SPRI beads (MagBio Genomics) and subsequent bisulfite conversion using the EZ DNA Methylation-Lightning Kit (Zymo Research) per the manufacturer's protocol. Bisulfite conversion efficiency averaged 99.4% as estimated by the measured percent of unmethylated CpGs in λ-bacteriophage DNA (New England BioLabs N3013S) added at a 1:200 ratio to each sample. Libraries for Illumina-based sequencing were prepared with the Pico Methyl-Seq Library Prep Kit (Zymo Research) using Illumina TruSeq DNA methylation barcodes. Libraries were run on a High Sensitivity chip using an Agilent TapeStation 4200 to assess size distribution and overall quality of the amplified library. Fluorometric quantitation and TapeStation size distribution estimates permitted equimolar pooling, and six pooled libraries per run were sequenced on an Illumina NextSeq 500 instrument using the NextSeq 500/550 V2 High Output reagent kit (1 x 75 cycles).