|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on May 05, 2023 |
Title |
FACS sorted GFP-RBM3 Low, day 17 [N9_1_low] |
Sample type |
SRA |
|
|
Source name |
BOB Cas9 clone 2
|
Organism |
Homo sapiens |
Characteristics |
cell line: BOB Cas9 clone 2 cell type: induced NGN2 Neurons treatment: Tunicamycin
|
Treatment protocol |
Incubation at 32°C for 72h
|
Growth protocol |
iPSCs were enzymatically detached and dissociated into single cells using Accutase and plated into GelTrex (1:100 dilution)-coated culture plates in TeSR-E8 medium supplemented with 10 µM Rho-associated protein kinase (ROCK) inhibitor. After 24 hours (day 1), TeSR-E8 medium was changed to DMEM/F12 medium with GlutaMAX, supplemented with 1x N-2 supplement, 1x Non-Essential Amino Acids, 50 nM 2-Mercaptoethanol, 100 U/mL Penicillin-Streptomycin, and 1 µg/mL Doxycycline Hyclate (Dox) (iN-1 medium). After 24 hours (day 2), the medium was replaced with the same medium as the previous day. From day 3 to day 6, the culture was fed daily with Neurobasal medium supplemented with 1x B-27 supplement (minus vitamin A), 1x GlutaMAX, 50 nM 2-Mercaptoethanol, 100 U/mL Penicillin-Streptomycin, and 1 µg/mL Dox, 10 ng/mL Neurotrophin-3 (NT-3), and 10 ng/mL Brain-derived neurotrophic factor (BDNF) (iN-2 medium). After day 6, the medium was changed every other day.
|
Extracted molecule |
other |
Extraction protocol |
Both clones of GFP-RBM3 iPSCs (N8, N9) were differentiated in GelTrex-coated T75 flasks. The total number of i-neurons used for each clone in each replicate was approximately 60,000,000 to ensure sufficient copies of individual sgRNAs could be obtained from the pool. 4 days post differentiation, the cultures were transduced with a whole-genome sgRNA lentiviral library (399 non-targeting sgRNAs and 91138 sgRNAs targeting 18466 genes across the human genome), co-expressing a BFP fluorescent reporter to indicate successfully transduced cells. The viral titer was determined in pilot experiments, resulting in 20% of the i-neurons becoming BFP-positive on day 18 to minimise multiple sgRNA entries into a single cell. I-neurons at 14, 15 or 16 days post differentiation (or 11, 12, 13 days post-transduction) were cooled at 32°C for 72h before dissociation with 20 min Accutase and 10 min Trypsin-EDTA incubation, followed by fluorescence-activated cell sorting (FACS). GFP intensity of all BFP-positive cells was manually separated into 4 quartiles, and only cells with the GFP intensity falling in the top or the bottom quartile were collected in separate tubes and their genomic DNA was extracted (see Genomic DNA extraction) immediately after. Around 3,000,000 i-neurons were collected in the low GFP or high GFP tube for each clone in each replicate. The sequencing library was created in a two-stage PCR reaction. The first stage amplifies the enriched gRNA cassettes: 2 μg DNA, 1.5 μL Forward Primer (10 μM), 1.5 μL Reverse Primer (10 μM), 25 μL NEB Q5 High-Fidelity 2x Master Mix in a 50 μL reaction. The PCR condition was: 98C for 30 sec, 25 cycles of (98C for 10 sec, 62C for 30 sec, 72C for 15 sec), and 72C for 2 min. PCR product was purified using Ampure XP Beckman magnetic beads and diluted to 200 pg/μL. The second PCR attached the Illumina adaptors and barcodes: 1 μL PCR product from the first reaction, 0.75 μL Forwards Primer (20 uM), 0.75 μL Reverse Primer (20 uM), 10 μL Roche 2X KAPA HiFi ReadyMix in a 20 μL reaction. The PCR condition was: 95C for 3 min, 9 cycles of (98C for 20 sec, 66C for 15 sec, 72C for 20 sec), and 72C for 1 min. PCR products were purified using Ampure XP Beckman magnetic beads and eluted in 35 μL TE buffer. PCR products were quantified with NEBNext Library Quant Kit for Illumina. The library was run on an Illumina NextSeq 550, using an Illumina 75 cycle, high output kit at 1.4 pM.
|
|
|
Library strategy |
OTHER |
Library source |
other |
Library selection |
other |
Instrument model |
NextSeq 550 |
|
|
Data processing |
bcl2fastq to assemble short reads count gRNAs according to gRNA library for each sample merge count vectors Assembly: N/A gRNA target genomic regions according to GRCh38 Supplementary files format and content: rbm3-tun-library.csv contains the gRNA vectors for the experiment
|
|
|
Submission date |
Nov 11, 2022 |
Last update date |
May 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 |
|
|
Platform ID |
GPL21697 |
Series (1) |
GSE217789 |
Whole-genome CRISPR screen to identify regulators of cold-shock protein RBM3 |
|
Relations |
BioSample |
SAMN31693724 |
SRA |
SRX18238817 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
|