GSM5618931: H2B-TKO;unc-85(cas1100)-rep1; Caenorhabditis elegans; ChIP-Seq
Synchronized young adults were harvested and washed 3 times with M9 to remove excess bacteria. Using 200 μl pipette tip, drip worm mix into a 100 ml beaker that contains liquid nitrogen to make frozen worm “popcorn”. Grind worms into a frozen powder with mortar and crosslinked with 1.1% formaldehyde for 15 min at room temperature, then formaldehyde was quenched by addition of glycine and the fixed powder was washed by cold PBS 3 times. The fixed powder was resuspended and homogenized 2-3 strokes using a glass Dounce homogenizer that is assumed to gently disaggregate cells. Nuclei preparation and chromatin digestion were conducted with SimpleChIP® Enzymatic Chromatin IP Kit (CST,#9003). Briefly, resuspend cells using buffer A and incubate on ice for 10 min; Pellet nuclei was collected and resuspended with buffer B, add Micrococcal nuclease and incubate for 4-12 min at 37°C to digest DNA to the length of approximately 150-900 bp, stop digestion by adding EDTA. Then samples were resuspended in ice-cold FA buffer with 0.1% SDS, and sonicated using a Bioruptor sonication system at 70% power for 20 cycles of 30 s on and 30 s off. The sonicated samples were then centrifuged at 4°C for 15 min at 16,000g and transfer supernatant to a new tube. Then, the cross-linked chromatin concentration was measured by BCA Protein Assay Kit (TIANGEN,#PA115) and and 2% of the extract was saved as the input sample. 5 μg chromatin were precleaned with protein A/G agarose beads. Add 10 ng Drosophila line 2 (S2) cells spike-in chromatin (Active Motif, Catalog No. 53083) to each sample, and immunoprecipitated overnight using 2 ug H3 antibody (Abcam, AB195277) and 0.5 ug spike-in antibody (Active Motif, Catalog No. 61686). Add 30 µl protein A/G agarose beads to each ChIP sample and rotate at 4°C for 2 h. 150 µl ChIP Elution Buffer was added and vortex gently at 1200 rpm for 30 min at 65°C. Reverse crosslink the samples with proteinase K. DNA were purified using QIAquick PCR Purification Kit (Catalog No. 28106), According to the manufacturer, we used 20 ng purified DNA samples to construct the ChIP–seq library, DNA library construction was conducted with VAHTSTM Universal DNA Library Prep Kit for Illumina® V3 (Vazyme, ND607).