 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 27, 2024 |
| Title |
ChIPseq NGN2 48h AroLITE Rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
ZIP13K2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: ZIP13K2
age: 48h
chip antibody: anti-FLAG antibody Merck, F1804
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were detached with Accutase solution (Sigma), washed twice in PBS and fixed in rotation with 1% formaldehyde for 10 minutes at room temperature. Subsequently, the reaction was quenched by addition of glycine leading to a final concentration of 125 mM. Per replicate, three million cells were used as starting material. In brief, we followed the ChIPmentation protocol version 3 for histone marks and transcription factors (Schmidl et al., 2015). Cells were lysed in lysis buffer 3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% N-laurosylsarcosine) supplemented with 1x cOmplete protease inhibitor cocktail. Afterwards, chromatin was sonicated for 10 minutes using a Covaris E220evolution focused-ultrasonicator with 2% duty cycles, 105 W peak incident power and 200 cycles per burst. Lysates were clarified by centrifugation for 10 minutes at 20,000g. 10% of the clarified lysate was put aside as input control. The remaining lysate was mixed with 50 µL of equilibrated anti-FLAG antibody (Merck, F1804, 1 µg total) coupled to DynabeadsTM Protein G magnetic beads (Invitrogen) and incubated on a 3D-shaker over night at 4oC. The next day, samples were washed twice in TF-wash buffer I (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Triton-X-100 and 2 mM EDTA pH 8.0) followed by two washes in TF-wash buffer III (10mM Tris-HCl pH 8.0, 250 mM LiCl, 1% Triton-X-100, 0.7% sodium deoxycholate and 1 mM EDTA pH 8.0) and a final wash with 10 mM Tris-HCl pH 8.0
All samples were tagmented for 5 minutes at 37oC using the Illumina Tagment DNA kit and immediately put on ice. Tagmented chromatin was washed in ice-cold wash buffer I and TET buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, 0.2% Tween-20) twice each, and reverse-crosslinked for 1h at 55oC and 9h at 65oC in the presence of 300 mM NaCl and proteinase K (Ambion). Subsequently, DNA was purified using AMPureXP beads. Sequencing libraries were amplified using the Kapa HiFi HotStart Ready Mix (Roche) and Nextera custom primers (Illumina) (Buenrostro et al., 2013) for a total of 12 cycles
Paired-end 100 with 50 million fragments per library
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
ChIP-seq
|
| Data processing |
Reads were mapped to genome using BWA and samtools was used for SAM to BAM conversion, sorting and indexing. Genome Analysis Toolkit v4 was used to remove duplicated reads. Peak calling was then performed using MACS3 v3.0.0 b1 using the input of the respective sample
genome_build: hg38
|
| |
|
| Submission date |
Nov 04, 2022 |
| Last update date |
Feb 27, 2024 |
| Contact name |
Alexandre P Magalhaes |
| E-mail(s) |
magalhae@molgen.mpg.de
|
| Organization name |
Max Planck Institute for Molecular Genetics
|
| Street address |
Ihnestrasse 63-73
|
| City |
Berlin |
| ZIP/Postal code |
14194 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE201655 |
An activity-specificity trade-off encoded in human transcription factors |
|
| Relations |
| BioSample |
SAMN31610382 |
| SRA |
SRX18173785 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6710859_ZIP13K2_NGN2_48h_AroLITE_3_peaks.narrowPeak.gz |
184.8 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
