|
| Status |
Public on Dec 13, 2022 |
| Title |
JM8.N4 mESC, Triptolide 4hr, RNAPolIIXChIP, rep2 |
| Sample type |
SRA |
| |
|
| Source name |
JM8.N18
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell line: JM8.N18
cell type: Mouse embryonic stem cell
spike-in reference_organism: Homo sapiens
spike-in cell_line: HEK293T
chip antibody: RPB1
antibody info: Cell Signaling Technology (#14958)
genotype: WT
treatment: Triptolide
time: 4 hours
|
| Treatment protocol |
Transcription inhibition was performed by adding triptolide to the media of confluent cells at 1 µM for 45 minutes.
|
| Growth protocol |
Mouse embryonic stem cells (JM8.N4 mESCs) were cultured on plates coated with 0.1% gelatin solution (Sigma-Aldrich) under feeder-free conditions in medium consisting of KnockOut DMEM (Thermofisher) with 15% FBS (HyClone, SH30396.03, Lot. No. AE28209315) and 1000U/mL LIF (home-made), 1 mM MEM Non-Essential Amino Acid Solution (Thermofisher), 2 mM GlutaMAX (Thermofisher), 100 μg/ml Penicillin-Streptomycin (Thermofisher), and 0.1 mM 2-mercapoethanol (Sigma) supplemented with 2i, 10 μM MEK inhibitor (Tocris) and 3 μM GSK inhibitor (Sigma). mESCs were fed daily by replacing half the medium and passaged every two days with 0.05% Trypsin-EDTA (Thermo Fisher Scientific). 1 day prior to harvesting, cells were swapped to medium as described above without 2i.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 10 mins. Following quenching, chromatin was sonicated. Chromatin crosslinked to RPB1 was immunoprecipitated and then reverse crosslinked. Naked DNA was further sonicated to the 400-800bp range.
DNA from RPB1 ChIP or input chromatin was used to prepare libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB), using NEBNext Multiplex Oligos for Illumina for indexing samples. Libraries were amplified with a number of PCR cycles according to the manufacturer's protocol on the thermocycler using KAPA HiFi HotStart ReadyMix (Roche) and purified twice using AMPure XP beads (Beckman Coulter). Purified libraries were quantified using qPCR. Following qPCR quantification, libraries were submitted for sequencing.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Basecalls were performed using bcl2fastq v2.20.0.422 for NovaSeq output.
Paired end reads were aligned to the a concatenation of the UCSC mm39 and hg38 genomes using bowtie2 v2.3.5.1 with --no-mixed --no-discordant.
Uniquely aligned reads were filtered for PCR duplicates using Sambamba markdup. Reads for each condition were randomly subsambled using normalization factors based on hg38 spike-in. Normalization factors were corrected using the ratio of human to mouse read counts in corresponding input samples.
Assembly: mm39; hg38
Supplementary files format and content: bigwig file with data based on all uniquely aligning non-duplicate reads
|
| |
|
| Submission date |
Dec 12, 2022 |
| Last update date |
Dec 13, 2022 |
| Contact name |
Viraat Y Goel |
| E-mail(s) |
viraat@mit.edu
|
| Organization name |
Massachusetts Institute of Technology
|
| Department |
Biological Engineering
|
| Lab |
Anders Hansen
|
| Street address |
Building 56 Room 787B, 32 Vassar Street
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL25526 |
| Series (1) |
| GSE207225 |
Ultra-deep 3D genome structure mapping with Region Capture Micro-C |
|
| Relations |
| BioSample |
SAMN32168073 |
| SRA |
SRX18646737 |
