GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1119601

Query DataSets for GSM1119601

Status

Public on Jun 05, 2013

Title

RevErb WT BMDM, H3K9ac ChIPseq

Sample type

SRA

Source name

bone marrow dervied macrophages

Organism

Mus musculus

Characteristics

cell type: bone marrow derived macrophages (RevErbalpha flox/flox; RevErbbeta flox/flox)
antibody: anti-H3K9ac (Millipore, 07-352)
genetic background: C57Bl6 (RevErbalpha flox/flox; RevErbbeta flox/flox)

Growth protocol

mouse bone marrow were differentiate to macrophages in RPMI medium containing 30% L-cell condition medium and 20% FBS. After differentiation, bone marrow derived macrophages were grown in 10% RPMI supplement with 10ng/mL M-CSF. RAW macrophages were cultured in RPMI with 10% FBS.

Extracted molecule

genomic DNA

Extraction protocol

ChIP-seq: Lysates were clarified from sonicated cells and protein-DNA complexes were isolated with antibody or with streptavidin-magnetic beads for biotinylated protein. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (minus exo) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, ChIP DNA was PCR-amplified with Illumina Genomic adaptors or with NEXTflex DNA barcodes adaptors for 18 cycles and library fragments (ChIP) (insert plus adaptor and PCR primer sequences) were size-selected (150-250bp) from a 2% agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on a Genome Analyzer II (Illumina) or HiSeq 2000 (Illumina) following the manufacturer's protocols.
GRO-seq: GRO-Seq was performed as described (Core, L.J., Waterfall, J.J., and Lis, J.T., Science 322, 1845-1848 (2008)) with minor modifications. Approximately 10 million nuclei per sample were extracted and used for run-on and BrU incorporation. BrU labeled nascent transcripts were immunoprecipiated with anti-BrU agarose beads (Santa Cruz Biotech), washed, eluted and precipitated in ethanol. BrU precipitated RNA were subjected for first strand complementary DNA synthesis. First, polyA tailed was added using Poly(A)-polymerase (NEB). Reverse transcription was then performed using Scriptscript III Reverse Transcriptase (Invitrogen) with 5' phosphorylated primer containing an abasic dSpacer furan with binucleotide barcode. Excess primers were removed by Exonuclease I (Fermentas). First-strand cDNA products were fragment with basic hydrolysis and size-selected (105-400 nt) in a 10% polyacrylamide TBE-urea gel (Invitrogen). cDNA was subsequently circularlized using CircLigase (Epicentre), and relinearized at the abasic dSpacer furan with ApeI (NEB). The ssDNA template was amplified to generate DNA for sequencing by Phusion Hig-Fidelity DNA Polymerase (Thermo Science). PCR product was purified and size selected (140-225 bp) by gel electrophoresis on a non-denaturing 8% polyacrylamide TBE gel (Invitrogen). Purified DNA was then sequence on Illumina Genome Analyzer II according to the manuactuere’s instructions.
5'GRO-seq: For each RAW264.7 cell line, 20x106 nuclei were prepared from approximately 30x106 cells. Nuclear run-ons were performed in parallel on 100 μl aliquots containing 5x106 nuclei as for conventional GRO-Seq. Reactions were stopped and RNA was extracted with 450 μl TRIzol LS reagent (Invitrogen) each according to the manufacturer’s instructions. Following DNase treatment, the RNA was hydrolyzed in 20 μl total volume with 2 μl RNA fragmentation buffer (Ambion) for 10 minutes, and divalent cations were removed by gel filtration. Fragmented RNA was then 3’-dephosphorylated with polynucleotide kinase (Enzymatics), for 2 h at 37°C. The reaction was stopped with EDTA and PNK was inactivated and RNA denatured by heating the reaction to 75°C for 5 minutes, then cooled on ice for 2 minutes. BrdU-containing RNA fragments were precipitated using anti-BrdU agarose beads. The resulting RNA was dephosphorylated with calf intestinal phosphatase (NEB) and 5’-de-capped with tobacco acid pyrophosphatase (Epicentre). The reaction was stopped and RNA was extracted with Trizol LS, and libraries were prepared by ligating Illumina TruSeq-compatible adapters to the RNA 3’ and 5’ ends with truncated mutant RNA ligase 2 (K227Q) and RNA ligase 1 (NEB), respectively, followed by reverse transcription, cDNA isolation, and PCR amplification for 12 cycles. Final libraries were size-selected on PAGE/TBE gels to 60-110 bp insert size. A detailed protocol is available on request.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

For ChIP-Seq and GRO-Seq samples (single end reads), reads were aligned to the mm9 genome using bowtie (v0.12.7), keeping only reads that mapped best to a single, unique location. Aligned read files were analyzed with HOMER (http://biowhat.ucsd.edu/homer/) to find peaks, calculate RPKM from the gene bodies of RefSeq genes, and perform other analyses in the study.
Genome_build: mm9
Supplementary_files_format_and_content: Processed files include BEDGRAPH (principle component values along the genome for Hi-C), text files (for all common, H3K4me1hi H3K4me3lo, and H3K4me1hi H3K4me3lo intergenic RevErb sites), and summary file (Signal for ChIPseq, GROseq, and 5'GROseq at RevErb bound peaks). All genomic coordinates are relative to mm9 (NCBI 37) mouse assembly.

Submission date

Apr 09, 2013

Last update date

May 15, 2019

Contact name

Michael T Lam

E-mail(s)

mtlam@ucsd.edu

Organization name

University of California, San Diego

Department

School of Medicine