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Series GSE217789 Query DataSets for GSE217789
Status Public on May 05, 2023
Title Whole-genome CRISPR screen to identify regulators of cold-shock protein RBM3
Organism Homo sapiens
Experiment type Other
Summary Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 induction during cooling remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator.
 
Overall design An RBM3 CRISPR knockout pooled screen is performed using two clones of GFP-RBM3 human iPSC-derived neurons (i-neurons). 4-day differentiated iPSCs are transduced with a custom-made whole-genome sgRNA lentiviral library, targeting critical exons of 18466 genes across the human genome, co-expressing a BFP reporter. Day 14-16 i-neurons are incubated at 32°C for 72h before dissociation and fluorescence-activated cell sorting (FACS) on day 17-19. BFP-positive cells with the highest and lowest GFP-RBM3 expression, which falls in the top and bottom quartile of GFP intensity profiles, are separately collected, denoted as high GFP and low GFP populations. Genomic DNA is sequenced to identify sgRNAs enriched in high or low GFP populations.
 
Contributor(s) Patikas N, Lin JQ, Pavlou S, Field SF
Citation(s) 37248947
Submission date Nov 11, 2022
Last update date Aug 05, 2023
Contact name Emmanouil Metzakopian
E-mail(s) em698@cam.ac.uk
Organization name UK Dementia Research Institute
Street address Addrenbookes Campus
City Cambridge
ZIP/Postal code CB20AH
Country United Kingdom
 
Platforms (1)
GPL21697 NextSeq 550 (Homo sapiens)
Samples (12)
GSM6726715 FACS sorted GFP-RBM3 High, day 17 [N8_1_high]
GSM6726716 FACS sorted GFP-RBM3 Low, day 17 [N8_1_low]
GSM6726717 FACS sorted GFP-RBM3 High, day 18 [N8_2_high]
Relations
BioProject PRJNA900543

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Supplementary file Size Download File type/resource
GSE217789_hsLibV1FullV1Nik_20nt.csv.gz 1.2 Mb (ftp)(http) CSV
GSE217789_rbm3-tun-library.csv.gz 2.3 Mb (ftp)(http) CSV
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Raw data are available in SRA
Processed data are available on Series record

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