title: GSM2729813: monocyte sample replicate 2; Bos taurus; Bisulfite-Seq
library_construction_protocol: One straw of Holstein bull semen was used for DNA extraction. After thawing at 37°C, the semen was washed with PBS to remove the extender, and incubated overnight at 55°C in 200 µl of lysis buffer (10 mM Tris-HCl pH 7.5, 25 mM EDTA, 1% SDS, 75 mM NaCl, 50 mM DTT and 0.5 µg glycogen) in the presence of 0.2 mg/ml proteinase K. After incubation with 25 µg/ml RNAse A 1h at 37°C, genomic DNA was twice extracted using phenol and phenol:chloroform (1:1), ethanol precipitated and washed. The dried pellet was resuspended in TE buffer (10 mM Tris HCl pH7.5, 2 mM EDTA). DNA extraction from fibroblasts and monocytes was performed as described (Kiefer H. (2015) Methods Mol Biol 1222, 267-80), except that cells were lysed by the direct addition of lysis buffer and proteinase K to the cell pellet. RRBS libraries were prepared as described ( Gu H., Smith Z.D., Bock C., Boyle P., Gnirke A. & Meissner A. (2011). Nat Protoc 6, 468-81; Auclair G., Guibert S., Bender A. & Weber M. (2014). Genome Biol 15, 545) from two Holstein sperm samples, two monocyte samples and two fibroblast samples. Briefly, 200 ng of genomic DNA were digested by MspI (Thermo Scientific), end-repaired and ligated to 55 bp Illumina adapters for paired-end sequencing. Size selection by gel excision was performed in order to select the fragments ranging from 150 to 400 bp (genomic fragments of 40-290 bp + adapters). The DNA was then purified using the MinElute gel extraction kit (Qiagen) and bisulfite-converted twice consecutively with the EpiTect bisulphite kit (Qiagen), following the manufacturer’s instructions for DNA extracted from FFPE tissues. Converted DNA was amplified with the Pfu Turbo Cx hotstart DNA polymerase (Agilent) using 14 PCR cycles for sperm and fibroblasts and 12 cycles for the monocytes. The libraries were then purified using Agencourt Ampure beads (Beckman-Coulter) and sequenced on an Illumina HiSeq2500 sequencer to produce 75 bp paired-end reads (Integragen SA, France).
attributes.tag: GEO Accession
attributes.value: GSM2729813