|
| Status |
Public on Feb 15, 2016 |
| Title |
DamIP-seq in IMR90hTert cells expressing EGFPB_DamK9A, (SS) rep2 |
| Sample type |
SRA |
| |
|
| Source name |
immortalized fetal lung fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
experiment: DamIP-seq
cell line: IMR90hTert
passage: 6-13
expressing: EGFP-DamK9A
treatment: Cells serum starved (8 hrs)
damip antibody: anti-N6-methyladenosine antibody (Millipore, catalog# ABE572, lot# 2377410)
|
| Growth protocol |
IMR90hTert cells were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DamIP-seq was performed as described (Xiao et al., 2010, 2012) with modifications. In brief, 10 µg of gDNA were sheared to 100-300bp with a Covaris S220 Focused-Ultrasonicator in 6x16mm µTUBE (Covaris, Cat. 520045). DNA was boiled 10’ at 98°C, cooled 10’ in ice, diluted with 1 volume of ice-cold 2X DamIP buffer (20 mM Tris-Cl pH 7.4, 300 mM NaCl and 0.2% NP40) and incubated O/N at 4°C under rotation with 4 µg of Anti-N6-methyladenosine antibody (Millipore, 1 µg/µl, ABE572). Antibody-DNA complexes were pulled-down with 25 µl of Dynabeads protein-A (Life technologies, 10002D) by 1h incubation at room temperature. Beads were washed four times in 1X DamIP buffer and resuspended in 300 µl of Elution buffer (50 mM Tris-Cl pH 8, 10 mM EDTA and 0.5% SDS) with 40 µg of Proteinase K. After 3h incubation at 48°C, immunoprecipitated single stranded DNA (ssDNA) was extracted with phenol chloroform, ethanol precipitated and resuspended in 17 µl of EB buffer.
Libraries were prepared starting from single strand cDNA with the TruSeq RNA sample prep v2 kit (Illumina, RS-122-2001) according to manufacturer’s instructions, but using Nucleospin columns and NTC buffer when required instead of AmpureXP beads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
EGFP_pooled_DamIP_SS.bedgraph
EGFPB_DamIP_SS_rep2
|
| Data processing |
library strategy: DamIP-seq
Tags were first trimmed using Cutadapt
Alignment : Tags with perfect matches were directly extracted from the eland_extended mode of ELAND (v2e in the Illumina CASSAVA pipeline version 1.82) output.
Alignment : Tags with multiple matches were mapped onto the Human genome build hg19 using the fetchGWI software and allowing 500 repeats on the genome. The tags mapping more than once onto the genome were then attributed a weight corresponding to the number of times the tag was sequenced divided by the number of genomic matches.
Pol III scores were calculated for bins including the annotated gene body ±150bp as the mean log2 (IPtag count/INPUTtag count) of two replicates per condition.
For de novo enriched regions discovery, the whole genome was split into 400 bp bins and bin scores were calculated as the mean log2 (MAF1tag count/EGFPtag count) for each condition. Bins having 1) more than 8 tags in the MAF1-expressing clones and 2) a positive score, were considered as potentially occupied
Genome_build: UCSC Hg19
Supplementary_files_format_and_content: bedgraph
|
| |
|
| Submission date |
Oct 13, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicolo Riggi |
| Organization name |
CHUV
|
| Department |
Département de Pathologie Expérimentale
|
| Lab |
Institut universitaire de pathologie
|
| Street address |
Bugnon 25
|
| City |
Lausanne |
| State/province |
VD |
| ZIP/Postal code |
1011 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE73928 |
Genome-wide MAF1-dependent regulation of RNA polymerase III transcription [DamIP-Seq] |
| GSE73936 |
Genome-wide MAF1-dependent regulation of RNA polymerase III transcription |
|
| Relations |
| BioSample |
SAMN04160423 |
| SRA |
SRX1330992 |
