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Sample GSM6135252 Query DataSets for GSM6135252
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.
 
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
 
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

Supplementary file Size Download File type/resource
GSM6135252_293FT_RIPseq_Cas13d_FLAG_RIP.peak.bed.gz 99.5 Kb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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