title: GSM9309954: 377_R; Rattus norvegicus; Bisulfite-Seq
library_name: GSM9309954
library_construction_protocol: Skeletal muscle tissue derived from aged rats (12 ± 2 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 (1427 ± 426 ng/mg tissue, 2 ± 0 A260/A280 &  2 ± 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 7.22 ± 0.23. RREM (reduced-representation enzymatic methyl-seq) libraries were prepared using 100 ng of genomic DNA in conjunction with the NEBNext Enzymatic Methyl-Seq kit (New England Biolabs, Massachusetts, USA). Briefly, DNA samples were digested using MspI restriction enzyme followed by end-repair and adapter ligation. A two step enzymatic conversion is then performed by Oxidation of 5mC and 5hmC using TET2 and glucosylation of 5hmC using T4-BGT. Deamination of unmodified cytosines was subsequently performed using APOBEC. Converted samples were then uniquely barcoded and amplified by PCR prior to sequencing. Sequencing was performed on an Illumina NextSeq 2000 with P4 XLEAP 100-cycle sequencing kit, generating 50bp paired-end reads.