GSM6569608: LNCaP-Input; Homo sapiens; ChIP-Seq
Cell pellets were lysed and chromatin fragmented using a Covaris M220 Sonicator, and the Covaris truChIP Chromatin shearing kit (Covaris; 520154). Briefly, cell pellets were thawed on ice and resuspended in 1 ml of 1X lysis buffer, supplemented with protease inhibitors. Samples were incubated on ice for 10 minutes, with gentle vortexing every 2 min. Nuclei were collected by centrifugation at 1700 g for 5 min (at 4 degrees C). The nuclei pellet was then resuspended in 1 ml of 1X wash buffer (supplemented with protease inhibitors), and collected by centrifugation as before. Samples were then washed twice, using centrifugation as before, with 1 ml of complete shearing buffer. Pellets were resuspended in 1 ml of fresh shearing buffer and transferred into a cold 1 ml AFA tube (Covaris). Sonication conditions were as follows: PIP 75, duty factor 10%, CBP 200, 4 degrees C, 12 minutes.  To verify sonication, aliquots of time-zero and sonicated samples were treated with RNase A (30 min at 37 degrees C) and reverse crosslinked overnight with proteinase K via incubation at 65 degrees C. After reverse crosslinking, DNA was purified using QIAquick PCR Purification Kit (Qiagen;. 28104), according to the manufacturer's instructions. Shearing efficiency was verified via agarose gel electrophoresis. Chromatin immunoprecipitation was done using the MAGnify Chromatin immunoprecipitation system (Life Technologies, 49-2024), according to the manufacturer's protocol, diluting the sheared chromatin at a 1:3 ratio in the kit's dilution buffer and using three reactions per sample. Three micrograms of rabbit polyclonal antibody against Lamin B1 were used, per reaction (Abcam; ab16048). After pulldown and purification, DNA fragments were sonicated for a second time, using the following parameters in a Covaris M220 sonicator: 130 microliter AFA tube, 10 o C, PIP 50%, duty factor 20%, CPB 200, for 45 seconds. ChIP-seq libraries were constructed by Azenta/Genewiz using the Illumina TruSeq library preparation approach and paired end 150 bp sequencing was performed