GSM4002631: mES Mll2KO shCXXC1 H3K27me3 rep2; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K27me3
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
source_name
embryonic stem cell
cell type
embryonic stem cell
cell line
v6.5
shRNA/sgrna
shCXXC1
genotype/variant
Mll2KO
chip antibody
H3K27me3 (Homemade, #67)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
RNA was extracted using RNeasy mini kit (Qiagen) and RNAse free DNaseI (Sigma) was used to eliminate DNA contamination. For ChIPseq, ESCs were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin were sonicated using a E220 focused-ultrasonicator (Covaris). Sheared chromatin, 5 µg antibody, and 50 µl protein A/G beads (Santa Cruz) were used for each immunoprecipitation. Immunoprecipitated DNA were purified after washing, eluting, and reverse-crosslinking and submitted for library preparation. For PROseq, nuclei were isolated by Dounce homogenizer with loose pestle. Nuclei were subjected to nuclear run-on in the presence of biotin-11-ATP/GTP/CTP/UTP and Drosophila S2 spike-in nuclei. Biotinylated nascent RNA was purified by Dynabeads M-280 streptavidin. For mRRBS, genomic DNA was digested with MspI (New England BioLabs) before size selection of 100- to 250-bp fragments with solid-phase reversible immobilization beads (MagBio Genomics). DNA was bisulfite converted with the EZ DNA Methylation-Lightning Kit. For CRISPR screen sequencing, reads were aligned to the sgRNAs using Bowtie1. ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit