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| Status |
Public on Jul 26, 2021 |
| Title |
14 hour IP #3 |
| Sample type |
SRA |
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| Source name |
C2C12
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| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12
hours after inducing differentiation: 14
antibody used for ip: Apobec2 (polyclonal, produced in rabbit)
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| Growth protocol |
C2C12s were plated at ~70% confluence 12 hours prior to inducing differentiation (seed ~2x10^6 cells in 10cm plates) maintained in DMEM (ATCC, 30-2002) with 10%FBS. This was followed by media change to DMEM with 2% horse serum (Life Biotechnologies, 26050-088) to induce differentiation. The cells (~5x10^6 /10cm plate) were harvested at 24-hour or 34-hour after inducing differentiation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed on plate with 1% PFA in PBS for 10 minutes at RT. Glycine was added to a final concentration of 125mM. Cells were washed 2 times with 1x PBS with protease inhibitor cocktail (PIC, Roche, 11836170001). They were lysed on the plate with cold Farnham lysis buffer to ~10x10^6 cells /mL (5mM PIPES pH 8.0, 0.5% NP-40, 85mM KCl,1mM EDTA, PIC) and incubated rotating for 15min at 4°C . Lysates were scraped off the plates, pelleted and resuspended in LB2 (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM 128EDTA, 0.5 mM EGTA, PIC) and incubated rotating for 15 minutes at 4°C and then centrifuged. Pellets were resuspended to 5x10^7 cells/mL in LB3 (10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium-deoxycholate, 0.5% sodium lauroyl sarcosinate, PIC) until suspension was homogenized. Samples were then sonicated using Covaris ultrasonicator model S220 for 15 minutes with the following settings: 140W peak power, 5% duty, 200 cycles per burst. TritonX-100 to a final concentration of 1% was added to the samples. Samples were clarified by centrifugation at 20,000 g for 10 minutes at 4°C. The supernatant is the soluble chromatin extract. The soluble fragmented chromatin from ~2.5x10^7 was used for each IP. For each IP 100ul Dynabeads (Thermofisher anti-rabbit M280, 11203D) were mixed with 10ul polyclonal rabbit-APOBEC2 antibodies (gift from Alin Vonica MD, PhD) incubating overnight (~16 hours). A magnetic stand was used to separate beads from the lysate and beads were washed one time each with for 5min in: low salt wash (0.1%SDS, 1%Triton X-100, 2mM EDTA, 20mM Tris pH8, 150mM NaCl, PIC), high salt wash (0.1%SDS, 1% Triton X- 100, 2mM EDTA, 20mM Tris pH8, 500mM NaCl, PIC), lithium chloride wash (150mM LiCl, 1% NP-40, 1% NaDOC, 1mM EDTA, 10mM TrispH8, PIC), TE wash (10mM Tris-HCl pH8, 1mM EDTA, 50mM NaCl, PIC). Beads were resuspended in 52 ul of elution buffer (50mM Tris-HCl pH8, 10mM EDTA, 1%SDS) and incubated at 30min at 65°C while shaking to prevent beads from settling. The eluate was transferred to a new tube, inputs of the same volume were incubated for 8 hours at 65°C to reverse the crosslink. The samples were treated with RNAse (Roche, Cat. No. 11 119 915 001) for 1 hour at 37°C, and with Proteinase K for 2 hours at 55°C. Fragmented DNA was purified 129 with Ampure beads (Agencourt AMPure XP beads A63881) as per the manufacter’s instructions.
ChIP-Seq libraries were prepared using NEBNext Ultra DNA Library Prep Kit as per manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Chromatin Immunoprecipitated DNA
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| Data processing |
Single-end reads were aligned to mm10 genome using the subread function in the Bioconductor Rsubread (Liao et al., 2013)
bigWig files for visualization were generated from aligned reads using the Bioconductor rtracklayer (Lawrence et al., 2009) and GenomicAlignments packages (Lawrence et al., 2013).
Quality metrics for ChIP-Seq data were assessed using ChIPQC bioconductor package (Carroll et al., 2014)
Reads mapping to more than one genomic location were filtered prior to peak calling using Model-based Analysis of ChIP-Seq (MACS2) (Feng et al., 2011; Zhang et al., 2008) with duplicate filtering applied and input DNA sample as a control
Genome_build: mm10
Supplementary_files_format_and_content: bigWig file contains normalised ChIP-Seq signal across the genome. Useful for display of the normalized ChIP-Seq signal at their location in the genome in a track format for uploading into a genome browser such as the UCSC genome browser or Broad Institute's IGV.
narrowPeak - contains the Peak calls using MACS2 in narrow peak format
Summits - Peak call summits in BED format.
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| Submission date |
Jul 26, 2018 |
| Last update date |
Jul 26, 2021 |
| Contact name |
Nina Papavasiliou |
| E-mail(s) |
n.papavasiliou@dkfz-heidelberg.de
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| Organization name |
German Cancer Research Center (DKFZ)
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| Department |
Division of Immune Diversity (D150) Deutsches Krebsforschungszentrum
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| Lab |
Prof. Dr. Nina Papavasiliou
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| Street address |
Im Neuenheimer Feld 280
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| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE117729 |
A novel role for the conserved, orphan deaminase APOBEC2 [ChIP-seq] |
| GSE117732 |
A novel role for the conserved, orphan deaminase APOBEC2 |
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| Relations |
| BioSample |
SAMN09724702 |
| SRA |
SRX4473664 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3307824_GFPsh.14hr.rA2.3_withInput_GFPsh.14hr.inp_peaks.narrowPeak.gz |
22.0 Kb |
(ftp)(http) |
NARROWPEAK |
| GSM3307824_GFPsh.14hr.rA2.3_withInput_GFPsh.14hr.inp_summits.bed.gz |
12.4 Kb |
(ftp)(http) |
BED |
| GSM3307824_Sorted_GFPsh.14hr.rA2.3Normalised.bw |
211.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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