 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 10, 2019 |
| Title |
Alyref Input rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
Aly-IN-2
|
| Organism |
Homo sapiens |
| Characteristics |
transgenic construct: FLAG-Alyref
antibody: None
replicate: 2
cell line: HEK293T
|
| Treatment protocol |
Three 15-cm dishes were seeded per ChIP condition with 5 x 106 cells/dish. Near endogenous expression of the FLAG-tagged proteins was induced for 48 hours in the stable cell lines by addition of tetracycline at 5 ng/mL for Nxf1, and 10 ng/mL for Chtop, and Alyref. Protein-DNA complexes were cross-linked in vivo by incubating the cells with 20 mL PBS-formaldehyde (1%).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell pellets were lysed in ChIP Lysis Buffer 1 (50 mM HEPES-NaOH pH7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100, protease inhibitors SigmaFAST) and rotated at 4°C for 5 minutes. Nuclei were then pelleted via centrifugation (3000 x g, 5 minutes at 4°C), and resuspended in ChIP Buffer 2 (10 mM Tris-HCl pH 7.3, 200 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, protease inhibitors SigmaFAST), before rotation at room temperature for 10 mins. Nuclei were pelleted via centrifugation (1500 x g, 5 minutes at 4°C) and resuspended in ChIP Lysis Buffer 3 (10 mM Tris-HCl pH 7.3, 200 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, protease inhibitors SigmaFAST). Samples were sonicated using a Bioruptor (High, 20 x [30s-ON/30s-OFF]) to generate chromatin fragments of 250-300 nts, and cleared by centrifugation (16100 x g, 15 minutes, 4°C). Lysate concentrations were measured by Bradford assay, and equal concentrations of chromatin were incorporated into the IPs. IPs were carried out overnight at 4°C using 5 µg of FLAG antibody (Sigma). 100 µL blocked protein-G Dynabeads were then added to the samples and incubated for 2 hours at 4°C. Following incubation, beads were washed with 4 x 0.5 mL RIPA Wash Buffer (50 mM HEPES-NaOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP40, 0.7% Na-deoxycholate, 0.1% N-lauroylsarcosine) and once with 0.5 mL ChIP Final Wash Buffer (10 mM Tris-HCl pH7.3, 1 mM EDTA, 50 mM NaCl). Complexes were eluted by adding 200 µL of ChIP Elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS) and incubated at 65°C for 30 minutes. NaCl was added to a final concentration of 200 mM and cross-links were reversed overnight at 65°C. Samples were then treated with RNase A (0.2 mg/mL) for 2 hours at 37°C, followed by proteinase K (0.2 mg/mL) for 2 hours at 55°C. DNA was purified via phenol-chloroform extraction and ethanol precipitation, and then resuspended in water.
Library construction was using the standard ChIP-seq protocol of Novogene (Beijing, China)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Reads were adaptor and quality trimmed, removing bases with a Q<15 from the 5’ end of the read, Q<20 from the 3’ end of the read and also if the average quality in a 4 nts window falls below 15. Trimmed reads greater than 36 nts were retained.
Reads were mapped to hg19 using BWA MEM version 0.7.17-r1188 (http://bio-bwa.sourceforge.net/) with the options `-M –k 25`.
Alignments were filtered to remove reads mapping to more than one location, other non-primary alignments, alignments that are not “proper-paired” and alignments with an alignment score < 30. PCR duplicates were removed using Picard MarkDuplicates
Coverage tracks were generated using genomecov from Bedtools, scaling depths to counts per million, and converted to Bigwig using BedGraphToBigWig from UCSC Kent utils.
Genome_build: hg19
Supplementary_files_format_and_content: Bigwig files showing read depth in counts per million
|
| |
|
| Submission date |
May 10, 2019 |
| Last update date |
May 12, 2019 |
| Contact name |
Ian Sudbery |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Sheffield
|
| Department |
Molecular Biology and Biotechnology
|
| Lab |
Sudbery Lab for Computational Genomics
|
| Street address |
Firth Court, Western Bank
|
| City |
Sheffield |
| ZIP/Postal code |
S11 8HG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE113953 |
Co-transcriptional loading of RNA export factors shapes the human transcriptome |
| GSE130992 |
ChIP-seq of TREX complex proteins in HEK293T cells |
|
| Relations |
| BioSample |
SAMN11618259 |
| SRA |
SRX5817189 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3759265_Aly-IN-2.bw |
276.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
