title: GSM8807013: celMBD2_input_replicate1; Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: Worm droplets in PBS frozen in liquid nitrogen were grinded into fine powder, followed by fixing in 1.1% formaldehyde in PBS. The fixation was quenched and further lysis was performed by adding MNase buffer supplemented with 0.2% NP-40 and 0.5% sodium deoxycholate. DNA is digested with Mnase at 37°C for 20 minutes. Then chromatin was solubilized by adding EDTA and Triton X-100 to a final concentration of 10 mM and 1%, nutated at 4°C for 2 hr. Two volumes of cold 150 mM NaCl FA buffer supplemented with 1% Triton™ X-100 + 0.1% sodium deoxycholate + 0.1% sarkosyl was added, followed by sonication with a Covaris M220 model (duty factor 20%, average power 8, cycles per burst 200, 60 s per cycle, total 5 cycles) to liberate chromatin. Immunoprecipitation of GFP::3xFLAGMBD-2 was performed anti-FLAG antibody-conjugated magnetic beads (MCE Cat# HY-K0207) overnight at 4°C. 5% of the protein was stored as input. The antibody/protein/DNA complexes were then eluted by Elution buffer (1%SDS/0.1M NaHCO3). The beads were then magnetized and the eluate was retrieved. The eluate was further subjected to RNase A and proteinase K treatment. Reverse crosslink was performed in 0.2 M NaCl salt at 65°C overnight in the thermomixer. The DNA was purified with PCR Purification Kit (QIAGEN, Cat. 28106). The samples were submitted to the Centre for PanorOmic Sciences (CPOS, HKU LKS Faculty of Medicine) for library preparation with KAPA Hyper Prep Kit following manufacturer protocol.
identifiers.namespace: GEO
identifiers.id: GSM8807013_r1