|
| Status |
Public on Mar 12, 2024 |
| Title |
K562 cells, SF3B1 K700E, 4 days, replicate 1 [LRSseq] |
| Sample type |
SRA |
| |
|
| Source name |
K562
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
cell type: Immortalized epithelial cells
genotype: SF3B1K700E
|
| Treatment protocol |
Modified allele expression induced with doxycycline (1ug/ml)
Cells (~20 million cells, per condition) were harvested at 4 days after doxycycline induction
|
| Growth protocol |
K562 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All extraction steps were performed on ice, and all buffers contained 25 uM α-amanitin, 40 U/ml RNase inhibitor, and protease inhibitor. Chromatin fractions were isolated from the cellular pellets using a previously published protocol. Chromatin was immediately dissolved in PBS and TRIzol Reagent. RNA was purified from chromatin using the RNeasy Mini kit according to the manufacturer’s protocol, including the on-column DNase I digestion. For genome-wide nascent RNA-seq, samples were depleted three times of polyA(+) RNA using the Magnetic mRNA isolation kit, each time keeping the supernatant, then depleted of ribosomal RNA using oligo probe-based rRNA depletion
A DNA adapter was ligated to 3′ ends of nascent RNA using the T4 RNA ligase kit by mixing adapter with nascent RNA. cDNA was generated from the adapter ligated RNA using the SMARTer PCR cDNA Synthesis Kit. cDNA was amplified by 15 cycles of PCR using the Advantage 2 PCR Kit with template switching custom oligo, cleaned up using a AMPure beads
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PacBio RS II |
| |
|
| Data processing |
library strategy: LRS-seq
Porechop v0.2.4 was used to remove external adapters, Cutadapt for removing 3’ adapters and Prinseq-lite to remove PCR duplicates.
Minimap2 was used to align to hg38 build.
Custom scripts in Python and R were used to generate Co-SE and cumulative distribution of 3’ RNAPII position
Assembly: hg38
Supplementary files format and content: tab-delimited text files include average/median 3'RNAPII position (for each gene) and co-transcriptional splicing efficiency (CoSE) values (for introns genome-wide) for each sample
|
| |
|
| Submission date |
Feb 23, 2023 |
| Last update date |
Mar 12, 2024 |
| Contact name |
MANOJ PILLAI |
| E-mail(s) |
[email protected]
|
| Organization name |
YALE UNIVERSITY
|
| Lab |
PILLAI LAB
|
| Street address |
300 GEORGE STREET #786
|
| City |
NEW HAVEN |
| State/province |
CT |
| ZIP/Postal code |
06511 |
| Country |
USA |
| |
|
| Platform ID |
GPL21311 |
| Series (2) |
| GSE225992 |
Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape [Long read sequencing] |
| GSE226003 |
Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape. |
|
| Relations |
| BioSample |
SAMN33427537 |
| SRA |
SRX19489864 |
