GSM4201614: set-32 mutant no RNAi input; Caenorhabditis elegans; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Adult
Cell type
Young adult
NA
NA
Attributes by original data submitter
Sample
source_name
Young adult whole animal
genotype
set-32(ok1457)
RNAi
no RNAi
chip antibody
input
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Synchronized young adult worms were first washed off the plates with M9 buffer. E. coli OP50 bacteria washed off together with the worms were separated and removed by loading the worms to 10% sucrose cushion and centrifuging for 1 minute at 600g in a clinical centrifuge. Worms were then pulverized by grinding in liquid nitrogen with a pre-chilled mortar and pestle and were stored at −80°C. Worm grinds from approximately 5000 worms were used for each chromatin immunoprecipitation experiment according to the procedure described in (Ni 2014). Anti-H3K9me3 (ab8898, Abcam) and anti-H3K23me3 (61500, Active Motif) antibodies were used for the H3K9me3 and H3K23me3 ChIP, respectively. Each ChIP experiments usually yielded 5–10 ng DNA. The entire ChIP DNA or 10 ng DNA in the case of ChIP input was used to make DNA library with the KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer's instruction. For each sample in a given assay worm grinds were thawed and subsequently crosslinked and sonicated to produce fragments between 200-500bp according to protocol described in (Ni, 2016). Samples were then used for ChIP (H3K23me1,2,3, H3K9me3, H3K27me3) or stored for library prep as input DNA. IP was performed with the following antibodies: anti-H3K9me3 (ab8898, Abcam), anti-H3K23me1 (39388, Active Motif), anti-H3K23me2 (39654, Active Motif), anti-H3K23me3 (61500, Active Motif), and anti-H3K27me3 (39535, Active Motif). For each antibody ~0.5-1.5% of input DNA was pulled down, with DNA yields between ~5-25 ng. 5 ng or less of DNA was used for library preparation using KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer's instruction. PCR was performed on library DNA for 12-17 cycles after which all libraries were pooled according to Illumina HiSeq specifications. Sequencing was sent to Illumina and carried out according to the following specifications: 50-nt single-end run, dedicated index sequencing. Dedicated 6-mer indexes were used to demultiplex the libraries of different samples.