title: GSM6585632: SSCT F2/M  bio rep 1; Mus musculus; Bisulfite-Seq
library_construction_protocol: Sperm was isolated from the cauda epididymis from control F1, SSCT F1, SSCT F2/M and SSCT F2/P offspring (n=5 for each). For the three SSCT F0 transplanted W/W-v males and DBA2/J controls, donor-derived DBA2/J and control epididymal sperm were isolated at 16-18 months of age, as they were needed to breed the offspring for a prolonged time. with sSperm was collected in an Eppendorf and washed in 500uL Phosphate-buffered saline (PBS). For purification of the sperm, the suspension was carefully pipetted into a falcon with 2mL PureSperm 70% (Nidacon) while having the falcon almost horizontal and centrifuged for 20 minutes at 300 RCF no break at room temperature (RT). The pellet was then washed with 5 mL HBSS or PBS and centrifuge for 10 minutes at 500 RCF after which the pellet was SNAP frozen and stored at -80ᵒC for further analysis. RRBS libraries were prepared using the Premium Reduced Representation Bisulfite Sequencing (RRBS) Kit (Diagenode Cat# C02030033). 100ng of genomic DNA were used to start library preparation for each sample, with the exception of samples 21, 23, 24 that had lower input. Following library preparation, samples were pooled together by 9 or 8. PCR clean-up after the final library amplification was performed using a 1.45x beads:sample ratio of Agencourt® AMPure® XP (Beckman Coulter). DNA concentration of the pools was measured using the Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific).  The profile of the pools was checked using the High Sensitivity DNA chip for 2100 Bioanalyzer (Agilent).  In case of too high adapter dimer peaks, the pools were size selected one more time using a 1.45x beads:sample ratio of Agencourt® AMPure® XP (Beckman Coulter) and quality control steps were performed again. RRBS library pools were sequenced on a HiSeq3000 (Illumina) using 50 bp single-read sequencing (SR50).