GSM5237109: mLN_GF_FSC_2 (WGBS); Mus musculus; Bisulfite-Seq
Genomic DNA was isolated from purified CD45‑CD31‑Ter119‑gp38+ using the AllPrep DNA/RNA Micro Kit (Qiagen, Cat. #80284) according to the manufacturer's instructions. Concentration and quality of the purified genomic DNA (gDNA) was determined by using NanoDrop (Thermo Fisher Scientific). Fragmentation of gDNA was carried out via Covaris S2 (Covaris), at duty cycle 10 %, intensity 4 and 200 cycles per burst during 80 sec, to obtain fragments with an average length of 300 bp. The size of the fragments was verified with Agilent Technologies 2100 Bioanalyzer. DNA sequencing libraries were generated from fragmented gDNA using the TruSeq DNA Sample Prep Kit v2 (Illumina, Cat. #15026486) according to the manufacturer's instructions and extending the workflow by adding one additional step: Subsequent to ligation of the adapter molecules to the DNA fragments, the sample was subjected to bisulfite conversion reaction using the EZ DNA Methylation Kit (Zymo Research, Cat. #D5001). The protocol for the True Seq DNA generation was then followed. The bisulfite-converted library was amplified by performing a PCR reaction (10 cycles, 98°C for 10 s, 63°C for 30 s, 72°C for 1 min) including the TruSeq primer mix and the KAPA Hifi Uracil+ Poly-merase Master Mix (Kapa Biosystems, Cat. #KK2801). The PCR product was purified and size controlled by Agilent Technologies 2100 Bioanalyzer (High Sensitivity DNA Chip). The libraries were sequenced on an Illumina HiSeq2500 sequencer using the TruSeq SBS Kit v3-HS (200 cycles, paired end run) with an average of 2*108 reads per replicate.