Sample information curated by ChIP-Atlas

Antigen

Antigen Class
RNA polymerase
Antigen
RNA polymerase II

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
WT (N2)
temperature
23˚C
generation
4th
repeat number
2
antibody
Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
100-200 μl frozen synchronized young adult worm pellets were used for each chromatin immnuoprecipitation experiment. Crushed pellets (pulverized by grinding in liquid nitrogen with a mortar and pestle) were resuspended in 1ml of pre-chilled RIPA buffer (1X PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1X HALT combined protease and phosphatase inhibitor cocktail [ThermoScientific]). To crosslink, formaldehyde was added to the crude extract to a final concentration of 2%. The lysate was rotated at 4°C for 10 minutes. 0.1 ml of 1M Tris-HCl (pH 7.5) was added to quench formaldehyde. Lysate was spun at 6000 x g for 30 second and the supernatant was removed. The pellet was resuspended in 0.5ml of pre-chilled RIPA buffer. The lysate was transferred to a 1.5 ml TPX tube (Diagenode) and sonicated (Bioruptor [Diagenode], output level: high, interval: 0.5, three times of 8-minute sonication). 50 μl of the lysate was used to make IP input library. For each immumoprecipiation experiment, 2 μg of anti-H3K9me3 (ab8898, Abcam) or anti-Pol II-S2 (ab5095, Abcam) was added to 400 μl of the lysate (containing approximately 50 μg DNA). The IP mix was rotated overnight at 4°C. 50 μl of Protein A Dynabeads (Life Technology) was added and rotated for another 2 hours. The beads were then washed three times with 800 μl ice-cold LiCl washing buffer (100 mM Tris-HCl, pH7.5, 500 mM LiCl, 1% NP-40 and 1% sodim deoxycholate). To elute the immunoprecipitation product and reverse crosslink, beads were incubated with 400 μl of worm lysis buffer (0.1 M Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, and 200 μg/ml of proteinase K) at 65 °C for 2 hours with agitation every 30 minutes (IP input lysate was treated similarly to reverse crosslink) and then subject to organic extraction and precipitation of DNA and RNA. DNA libraries were prepared using KAPA Hyper Prep Kits (KAPA Biosystems) according to the manufacturer's instructions

Sequencing Platform

instrument_model
Illumina HiSeq 2000

ce11

Number of total reads
4494469
Reads aligned (%)
96.0
Duplicates removed (%)
9.2
Number of peaks
3452 (qval < 1E-05)

ce10

Number of total reads
4494469
Reads aligned (%)
96.0
Duplicates removed (%)
9.2
Number of peaks
3456 (qval < 1E-05)

Base call quality data from DBCLS SRA