GSM2870886: 40H2 [BisPCRseq]; Mus musculus; Bisulfite-Seq
For DNA extraction, 1/4 of the extracted sperm was incubated overnight in 600 µl PK buffer (10 mM Tris-HCl, 100 mM NaCl, 25 mM EDTA, 1% SDS) with 2 µl Proteinase K enzyme (19 mg/ml) and 0.1M DTT at 55 °C with slow rotation. 750 µl phenol was added to the samples and agitated for several minutes before spinning at 17,000 x g for 5 mins at 4 °C. The upper aqueous phase was transferred to a new tube and the process repeated with phenol:chloroform, then chloroform alone. After the final spin, 5 µl Rnase was added and samples were incubated at 37 °C for 60 mins. 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% EtOH were added and samples incubated at -20 °C for 1 hour. Samples were spun at 17,000 x g for 10 mins at 4 °C and the pellet washed with 75% EtOH. Finally, the pellet was air-dried at 37 °C, resuspended in 200 µl TE buffer and incubated at 50 °C for 3 hours before storage at 4 °C. DNA from sperm was diluted to a concentration of 11 ng/µl and 45 µl of each sample was sent for sequencing to the Genome Centre Facility at Charterhouse Square, QMUL. Bis-PCR-Seq was performed using the 48.48 layout on the Fluidigm® C1 system (Fluidigm®, USA), coupled with Illumina MiSeq sequencing using version2 chemistry (150 bp, paired-end).
GEO Accession
GSM2870886