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| Status |
Public on Mar 20, 2024 |
| Title |
HEK293T cells, CTut |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T (stable cell line; non-targeting shRNA)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
treatment: non-targeting shRNA
antibody: Anti-G4 1H6 antibody (Sigma; MABE1126)
fraction: Chromatin
|
| Treatment protocol |
Samples 1 and 2 were processed for G4-quadruplex immunoprecipitation using the AbC G4-ChIP protocol. The corresponding pooled input was also processed (Sample 3). Sample 4 was processed using the conventional G4-quadruplex immunoprecipitation (PMID: 29470465) alongside the corresponding input (Sample 5). For samples 6 and 7, the genomic DNA were extracted from untransduced HEK293T cells. The sonicated genomic DNA were processed for in vitro G4-quadruplex immunoprecipitation directly (Sample 6) or after heat-denaturation and snap-chilling (Sample 7). The input (Sample 8) corresponding to the Samples 6 and 7 was the genomic DNA without any immunoprecipitation. Samples 9 and 11 were processed using AbC G4-ChIP without any transfection of control DNA (pGEMT Easy vector carrying an insert with G4-quadruplex-forming signature sequence). For samples 9 and 11, 500ng of each of the control DNA and a carrier DNA (vector carrying an insert devoid of G4-quadruplex-forming sequence signature) were first mixed, treated with 150 mM KCl, incubated with 1H6 antibody, crosslinked and added to the respective cell lysates for sonication and subsequent steps. Samples 10 and 12 were the inputs for samples 9 and 11 respectively. Samples 13 and 15 were transfected with 1.25 ug of each of control and carrier DNA mix per 10 cm dish using calcium-phosphate transfection method. Following 12 hours of transfection, the cells were processed for G4-quadruplex immunoprecipitation using AbC G4-ChIP. Samples 14 and 16 were the inputs for samples 13 and 15 respectively. From the step of reverse-crosslinking till elution, the inputs were treated the same way as the immunoprecipitated samples.
|
| Growth protocol |
HEK293T cells, transduced with non-targeting lentiviral (CT) or CGGBP1-targeting (KD) shRNA, as described elsewhere (PMID 31547883), were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution at 5% CO2 and 80% RH.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For all the G4-quadruplex immunoprecipitations, the cells were processed from two 10 cm dishes for each sample. The inputs were derived either from respective samples or by pooling from CT and KD samples. The immunoprecipitated DNA was purified using ethanol precipitation follwed by dissolution nuclease-free water.
Libraries were synthesized as follows:
(i) End-repair and pre-processing: As per the manufacturer’s instruction for Ion Express Plus Fragment Library kit (Cat. No. 4471269) from ThermoFisher Scientific for samples 1 to 3.
(ii) Sequencing library preparation: As per the manufacturer’s instruction for PCR sequencing kit (SQK-PSK004) from Oxford Nanopore Technologies for samples 4 to 16.
For samples 4 to 16, 4 – 8 ng of DNA were subjected to end-repair, ligation of PCR-adapters followed by fragment size selection using 0.4x AMPure XP beads. The adapter-ligated fragments were subjected to PCR amplification followed by size selection using 0.6x AMPure XP beads. The library was then subjected to sequencing adapter ligation followed by sequencing on MinION device using flow cells (R9.4.1). The mean length of the sequenced reads was in the range of 0.8 – 1 kb.
G4-quadruplex immunoprecipitation
Ion Torrent S5, ONT MinION Mk1B
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
MinION |
| |
|
| Description |
Sample 9
|
| Data processing |
For the samples 1 to 3, the raw sequenced reads were subjected to proprietary quality trimming (minimum Q20) for the IonTorrent platform. Quality-filtered reads with minimum length of 100 bases were used for further analysis. For the rest of the samples, the sequenced reads on the ONT platform were basecalled in real-time (minimum Qscore = 8) using guppy basecaller. The failed reads were subjected to basecalling in a locally installed guppy basecaller. The qualified reads were combined and subjected removal of sequencing adapters using Porechop with default options.
The reads were aligned using minimap2 against the unmasked hg38 (annotated chromosomes except chromosomes Y and M). The uniquely aligned reads were further analyzed for coverage on 0.2 kb genomic bins. Bins with coverage <3 and > 100 were eliminated for downstream analyzes.
Broadpeaks were called using MACS2 callpeak on the datasets against the inputs. A broadpeak q-value cutoff of 0.01 was used to call the peaks with a fixed background local lambda.
Assembly: hg38 (unmasked)
Supplementary files format and content: BED, broadpeaks discovered using MACS2 callpeak
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| |
|
| Submission date |
May 08, 2022 |
| Last update date |
Mar 20, 2024 |
| Contact name |
Umashankar Singh |
| Organization name |
IIT Gandhinagar
|
| Department |
Biological Sciences and Engineering
|
| Lab |
HoMeCell Lab
|
| Street address |
Acad Block 4 Room 317 IIT Gandhinagar Palaj
|
| City |
Gandhinagar |
| State/province |
Gujarat |
| ZIP/Postal code |
382055 |
| Country |
India |
| |
|
| Platform ID |
GPL24106 |
| Series (1) |
| GSE202456 |
G4 quadruplex landscape and its regulation revealed by a new antibody capture method |
|
| Relations |
| BioSample |
SAMN28155641 |
| SRA |
SRX15205619 |
