GSM9307849: GC-AS-10308-7R,Recovery,7,male; Homo sapiens; Bisulfite-Seq
Skeletal muscle tissue derived from humans (21 ± 3 mg) was added to 2 mL homogenizing mix tubes (Fisherbrand™, Fisher Scientific) containing 180 μl Buffer ATL (Qiagen, Hilden, Germany) before transferring to a tissue homogenizing instrument (MagNA Lyser; Roche, Basel, Switzerland) for subsequent homogenization (3 × 45 s at 6000 rpm, placing samples on ice for 5 mins between each disruption). Following homogenization, 20 μl proteinase K (Qiagen) was added and samples were incubated at 56°C until complete lysis (≈ 30-60 mins) before continuing with DNA purification using a DNeasy® Blood & Tissue kit (Qiagen) as per the manufacturer's instructions. DNA quantity and quality for both human (251 ± 47 ng/mg tissue, 2 ± 0 A260/A280 and 2.1 ± 0.1 A260/A230) samples were quantified using spectrophotometry (NanoDrop™ 2000; Thermo Fisher Scientific) and concentrations confirmed via fluorometry (Qubit®, Qiagen). DNA integrity was confirmed prior to sequencing via Agilent Bioanalsyer 2100 with DIN scores 7.11 ± 0.17. RRBS libraries were prepared from 100ng of genomic DNA using the Diagenode Premium RRBS v2 library preparation kit, as per the manufacturer's protocol. Two pools of 8 samples, and three pools of 7 samples were prepared after qPCR quantification of adapter-ligated sample and prior to bisulphite conversion. 14 cycles of final library amplification were performed after assessment of bisulphite converted library pool by qPCR. Libraries were sequenced using the Illumina NextSeq 2000 with P3 SBS 100-cycle sequencing kit, generating 50bp paired-end reads.