GSM3845594: Input March R1: Input DNA; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Embryo
Cell type
Embryonic liver
NA
NA
Attributes by original data submitter
Sample
source_name
Fetal liver
strain
C57BL/6
embryonic day
E14.5
tissue
Fetal liver
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
2x107 cells were washed with PBS once, then cross-linked with 1% formaldehyde for 10 minutes at room temperature. Cross-linking was quenched with 5M glycine for 1 minute at room temperature, and cells were washed with PBS once. Cells were incubated in swelling buffer for 20 minutes on ice, followed by dounce homogenization to isolate cross-linked nuclei. Nuclei were placed in lysis buffer for 30 minutes and then sonicated into ~200 bp fragments using a Diagenode Bioruptor. Samples were diluted and immunoprecipitated with an antibody to H3K4Me1 (Abcam #ab8895), H3K4Me3 (Active Motif # 39916), H3K27Ac (Active Motif # 39134), H3K4Me2 (Millipore # 07-030), or nonspecific rabbit immunogloblulin G (Millipore # 12-370) on a rotator for 18h at 4C. DNA-protein complexes were recovered with protein G magnetic beads (Invitrogen). Samples were cleaned with QIAquick PCR purification kit (QIAGEN) following manufacture's protocol. Libraries were prepared as previously described (Su, M.Y., Steiner, L.A., Bogardus, H., Mishra, T., Schulz, V.P., Hardison, R.C., & Gallagher, P.G.) Approximately 500ng of ChIP DNA was run on a 1.5% agarose gel to size select ChIP DNA in the 200-300-bp range. The size-selected ChIP DNA was purified using a gel extraction kit (Qiagen) and processed for sequencing as per manufacturer's protocol. Briefly, size-selected ChIP DNA was end repaired using an End-IT DNA end repair kit (ER0720; Epicenter), followed by addition of an adenine base at the 3′ end by Klenow reaction and Solexa adaptor ligation. The modified DNA was then PCR amplified with one initial heating step of 98°C for 30 s, followed by 15 cycles of amplification with a melting temperature of 98°C for 30 s, an annealing temperature of 65°C for 30 s, and a product extension at 72°C for 30 s. At the end of the amplification, a final extension at 72°C for 5 min was performed. Amplified ChIP DNA was then size selected on a 1.5% agarose gel, purified by using a gel extraction kit (Qiagen), and subjected to deep sequencing.