|
Status |
Public on Oct 09, 2023 |
Title |
293FT, RIPseq, Cas13d, FLAG, RIP |
Sample type |
SRA |
|
|
Source name |
human embryonal kidney cells
|
Organism |
Homo sapiens |
Characteristics |
tissue: kidney cell line: 293FT transfection: pLenti-Cas13d-nes-puro time: Day 3 after transient transfection rip antibody: FLAG (Invitrogen #MA1-91878)
|
Treatment protocol |
HEK293FT cells were plated in 15 cm plates and transfected at > 50% confluency with 25 µg of indicated plasmids using NeofectTM DNA transfection reagent according to the manufacturer’s protocol.
|
Growth protocol |
HEK293FT cells were regularly tested negative for mycoplasma contamination and maintained in DMEM (SEVEN Biotech) supplemented with 10% fetal bovine serum (ExCell) and 1% penicillin–streptomycin (Solarbio). Cells were passaged every 2-4 days to maintain exponential growth and were kept in a humidity-controlled incubator at 37°C with 5% CO2.
|
Extracted molecule |
total RNA |
Extraction protocol |
After 3 days transfection, cells were harvested by cross-linking with 0.3% formaldehyde for 10 min at room temperature and lysing with RIPA lysis buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitor and RNase inhibitor) for 10 min on ice before sonication. The supernatant was collected and incubated with IgG and FLAG antibodies at 4 °C overnight. After washing with RIPA buffer (50 mM Tris (pH 7.6), 1 M NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate), RNA was eluted from the beads with elution buffer (0.1 M NaHCO3 and 1% SDS, addition with proteinase K and RNase inhibitor) at room temperature for 10 min with gently vortex. The eluted material was then de-cross-linked at 65 °C for 45–60 min and treated with RNase inhibitor and proteinase K. Afterwards, RNA was purified using TRIzol LS reagent (Life Technologies) and treated with DNase I to remove any residual DNA. RIP RNA was used for library preparation.
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|
|
Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
|
|
Description |
Binding RNA
|
Data processing |
The RIP-seq reads were aligned against the hg38 human reference genome using HISAT2. Since the RIP-seq data is strand-specific, the peaks were called (RIP vs input) and identified for “+” and “–” strands separately using MACS2, with false-discovery rate (FDR) ≤ 0.01 and arbitrary extension size of 150 bp. Assembly: hg38 Supplementary files format and content: tab-delimited text files include peak called for each Sample
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|
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Submission date |
May 12, 2022 |
Last update date |
Oct 09, 2023 |
Contact name |
Teng Fei |
E-mail(s) |
feiteng@mail.neu.edu.cn
|
Phone |
86-24-83656103
|
Organization name |
Northeastern University, China
|
Department |
College of Life and Health Sciences
|
Lab |
Teng Fei's lab
|
Street address |
195 Chuangxin Rd. Life Sciences Building A426. Hunnan District.
|
City |
Shenyang |
State/province |
Liaoning |
ZIP/Postal code |
110169 |
Country |
China |
|
|
Platform ID |
GPL24676 |
Series (2) |
GSE202898 |
Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems [RIP-seq] |
GSE202899 |
Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems |
|
Relations |
BioSample |
SAMN28209168 |
SRA |
SRX15244508 |