Adult total nuclei (enriched for germ cell nuclei)
strain
FAS58
genotype
his-72(uge40[K27M]) III
chip antibody
H3K36me3 (Abcam #ab9050)
developmental stage
adult
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Native ChIP was performed as described previously (Steiner and Henikoff, 2014f) with minor modifications. Briefly, synchronized worm populations were grown on 15-cm plates seeded with NA22 bacteria. Worms were harvested as young adults, washed in M9 and resuspended in buffer A supplemented with 0.5% NP-40 and 0.1% Tx-100. Total nuclei, which are enriched for germ cell nuclei based on morphologic analysis, were obtained by light grinding of the worms under liquid nitrogen followed by douncing, low speed spin and washing of the nuclei. Chromatin was digested to mononucleosomes using MNase (NEB, #M0247S) at 37°C using 5 µl of enzyme per 150 µl of nuclei in 1000 µl of the buffer. Each sample was divided in 6 parts that were incubated for 3, 5, 8, 10, 12 and 15 minutes, respectively, and then pooled together in order to obtain nucleosomal ladders. Following the digestion, chromatin was extracted, solubilized and pre-cleared with empty beads. ChIP was performed overnight at 4°C from the soluble chromatin fraction using antibodies against H3K27me2/me3 (Active Motif #39535), OLLAS (Novus Biologicals #NBP1-06713B), H3.3K27M mutant (Milipore #C946H91) or H3K36me3 (Abcam #ab9050). ChIP was followed by 1 h incubation of antibody-bound chromatin with magnetic beads at 4°C (50 µl 1:1 mixture of Dynabeads Protein A and Dynabeads Protein G, Invitrogen). DNA from both input and IP samples was then extracted using phenol/chloroform. Libraries were prepared using NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads using 100 ng of input DNA. Libraries were sequenced using the TruSeq SBS HS v3 chemistry on an Illumina HiSeq 2500 sequencer. NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads