GSM2830111: DF3P25; Rattus norvegicus; ChIP-Seq
The epididymis was dissected free of connective tissue, a small cut made to the cauda and tissue placed in 3 ml of phosphate buffered saline (PBS) for 20 minutes at room temperature and then kept at 4ºC to immobilize the sperm. The epididymal tissue was minced and then the released sperm centrifuged at 11,000 x g and the pellet resuspended in NIM buffer and stored at -80ºC until processed further.  To isolate DNA, 100 µl of sperm suspension was combined with 820 µl DNA extraction buffer 1M Tris HC1, pH 8.0; 0.5M EDTA (ethylenediaminetetraacetic acid); 10% SDS (sodium dodecyl sulfate) and 80 µl 0.1M DTT (dithiothreitol). The sample was incubated at 65ºC for 15 minutes. Following this incubation, 80 µl proteinase K (20 mg/ml) was added and the sample incubated at 55ºC for at least 2 hours under constant rotation. Then 300 µl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A) was added, the sample mixed thoroughly and incubated for 15 min on ice. The sample was centrifuged at 13,500 rpm for 20 min at 4ºC. 1 ml of the supernatant was transferred to a 2 ml tube and 2 µl of Glycoblue (Invitrogen, Carlsbad, CA) and 1 ml of cold 100% isopropanol were added. The sample was mixed well by inverting the tube several times then left in -20ºC freezer for at least one hour. After precipitation, the sample was centrifuged at 13,500 rpm for 20 min at 4ºC. The supernatant was taken off and discarded without disturbing the blue pellet. The pellet was washed with 70% cold ethanol by adding 500 µl of 70% ethanol to the pellet and returning the tube to the freezer for 20 min. After the incubation, the tube was centrifuged for 10 min at 4ºC at 13,500 rpm and the supernatant discarded. The tube was spun again briefly to collect residual ethanol to bottom of tube and then a gel loading tip was used to remove as much liquid as possible. Pellet was air-dried at RT until it looked dry (about 5 min). Pellet was then resuspended in 100 µl of nuclease free water. Equal amounts of DNA from individual sperm samples were used to produce three DNA pools for histone chromatin immunoprecipitation (ChIP). The antibody used for the H3 location ChIP was Anti-Histone H3, Ct, pan, clone A3S, manufactured by Millipore (Cat. #05-928, Lot #2780491). The antibody used for the H3K27me3 ChIP was Anti-Trymethyl-Histone H3 (Lys27 polyclonal antibody) manufactured by Millipore (Cat. #07-449, Lot #2826067). The ChIP pools were used to create libraries for next generation sequencing (NGS) using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (NEB, San Diego, CA). The manufacturer protocol was followed. Each pool received a separate index primer. NGS was performed at the WSU Spokane Genomics Core using Ilumina HiSeq 2500 with a PE50 application, with a read size of approximately 50 bp and approximately 35 million reads per pool. Six libraries were run in one lane.
GEO Accession
GSM2830111