2024-08-26
ENA first public
ERC000011
ENA-CHECKLIST
Umeå universitet, Department of molecular biology
INSDC center name
public
INSDC status
ERS20894873
SRA accession
E-MTAB-14379:t0_PCR_3_S6
Submitter Id
ArrayExpress
broker name
not collected
collection date
t0_pcr_rep3
condition_name
Brunello-kinome
crispr library
Protocols: For each screen, 3 × 106 MiaPaCa-2 cells in 1 ml of DMEM medium with polybrene (8 μg/ml) were seeded per well of a 6-well plate and appropriately diluted amount of concentrated lentivirus in 180 μl of DMEM media was added in order to obtain 0.3 MOI (the viral titer was assessed before the screen as well as during the screen). After 24 h incubation, medium with virus was discarded, cells counted, re-seeded either on 150 mm culture dishes (24 × 106 cells) or 175 cm2 flasks (30 × 106 cells), and cultivated under puromycin selection (1 μg/ml) for 3 days. For initial screen with lentiCRISPRv2-GFPg1, 3 × 106 cells were initially infected per sample (n = 1) and cultivated for 2 weeks after puromycin selection. In case of a screen with Brunello kinome library, 1.0 × 107 cells  (1000× coverage) were initially infected per sample (n = 3) and further cultivated for 2 weeks after puromycin selection.    For each screen, 3 × 106 MiaPaCa-2 cells in 1 ml of DMEM medium with polybrene (8 μg/ml) were seeded per well of a 6-well plate and appropriately diluted amount of concentrated lentivirus in 180 μl of DMEM media was added in order to obtain 0.3 MOI (the viral titer was assessed before the screen as well as during the screen). After 24 h incubation, medium with virus was discarded, cells counted, re-seeded either on 150 mm culture dishes (24 × 106 cells) or 175 cm2 flasks (30 × 106 cells), and cultivated under puromycin selection (1 μg/ml) for 3 days.  For initial screen with lentiCRISPRv2-GFPg1, 3 × 106 cells were initially infected per sample (n = 1) and cultivated for 2 weeks after puromycin selection. In case of a screen with Brunello kinome library, 1.0 × 107 cells  (1000× coverage) were initially infected per sample (n = 3) and further cultivated for 2 weeks after puromycin selection.    For gDNA isolation either 3.0 × 107 cells (lentiCRISPRv2-GFPg1 and Brunello kinome library) or 3.0 × 108 cells (whole-genome Brunello library) per sample were harvested and processed according to Chen et al. 26. Briefly, cells were lysed in 50 mM Tris, 50 mM EDTA, 1% SDS, pH 8 in the presence of proteinase K (100 μg/ml) overnight at 55°C. Next day, samples were treated with RNAseA (50 μg/ml) for 30 minutes at 37°C and cooled on ice. After addition of 7.5M ammonium acetate, the clarified supernatant was further used for isopropanol precipitation of gDNA. Ethanol-washed gDNA pellets were dissolved in nuclease-free water and DNA concentration was assessed by Qubit. Libraries were prepared either using our CRISPR-MIP procedure (https://www.biorxiv.org/content/10.1101/2024.03.28.587082v1.full) or by traditional PCR (https://pubmed.ncbi.nlm.nih.gov/29224076/)
description
not collected
geographic location (country and/or sea)
not applicable
isolate
synthetic construct
organism
synthetic construct
organism
rep3
replicate
8
run_number
PCR
sampleprep_protocol
lib
sampletype
synthetic construct
scientific_name
NA
day
timepoint
t0_PCR_3_S6
title
gDNA
treatment