GSM2674831: Primary spermatocytes 2; Mus musculus; Bisulfite-Seq
Spermatocytes and spermatids were isolated from testes using 25ug/mL liberase TM (Roche) followed by elutriation and percoll gradient centrifugation. Caput, corpus, cauda, and vas deferens were dissected and genomic DNA was isolated from all samples by lysing in extraction buffer (4.24 M guanidine thiocyanate (Sigma), 100 mM NaCl (Ambion), 1% N-Laurylsarcosine (Sigma), 150 mM DTT (Promega) and 200 ug/mL Proteinase K (Sigma)) at 56C for 2 hours followed by isopropanol precipitation. Samples were then sonicated on a Covaris S-220 to achieve a median size of 200-250 base pairs. DNA fragments were cleaned up using phenol:choloroform:isoamyl alcohol and phase-lock tubes. 500 ng of DNA was used as input for 2 rounds of sodium bisulfite conversion using the EZ DNA Methylation – Lightning Kit (Zymo). Library construction was performed using TruSeq DNA Methylation Kit (Illumina) and Accel-NGS Methyl-Seq DNA Library Kit (Swift) according to the manufacturer’s instructions. For TruSeq (Illumina) libraries, 50ng bisulfite converted DNA was used and individual libraries were barcoded using the TruSeq DNA Methylation Index PCR Primers (IIlumina) to allow for multiplexed sequencing of libraries. Size selection and DNA purification was performed using Ampure XP beads (Agencourt) according to the TruSeq DNA Methylation kit protocol. For Accel-NGS Methyl-Seq (Swift) libraries, 30ng bisulfite converted DNA was used and barcoded following the (Swift) manufacturer’s instructions including 6 rounds of PCR. All final libraries were analyzed by Fragment Analyzer and quantitated using the Qubit as well as the KAPA Library Quantitation qPCR kit.  Bisulfite-Seq (Accel-NGS Methyl-Seq (Swift)) Bisulfite-Seq (Truseq)