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Sample GSM8527594 Query DataSets for GSM8527594
Status Public on Sep 25, 2024
Title WT_Input1_rep2
Sample type SRA
 
Source name blank sample
Organism blank sample
Characteristics screening library: Human Transporter KO (Addgene #213695)
Treatment protocol Cells were transduced with lentiviral particles containing KO library in triplicates and selected with blasticidin to remove non-transduced cells. After 13 days of selection, each replicatewas independently was passaged in standard growth media for a total of five weeks. Samples were harvested for NGS analysis 3 to 7 weeks after transduction.
Growth protocol HCT116 (RRID:CVCL_0291) cells and HCT116 KO clones were cultivated in RPMI 1640 (R8758 Sigma) supplemented with 10% Fetal Bovine Serum (10270-106, Lot 42F8381K, Gibco) and Penicillin-Streptomycin (15140-122, Gibco).
Extracted molecule other
Extraction protocol Genomic DNA was purified using QIAamp DNA Mini columns (Qiagen)
gDNA was digested overnight with 20U PacI (NEB) and double size-selected to enrich the sgRNA containing fragments (380 bp). Briefly, 0.7 x vol AmpliClean magnetic beads (Nimagen) were added to the sample to bind large gDNA fragments; the cleared supernatant was added to fresh beads (0.6 x volume of the original gDNA sample) to bind the smaller fragments. After size selection, sgRNA sequences were amplified in with Q5 Polymerase (NEB) using the program recommended by the manufacturer (28 cycles, 65°C annealing temperature). PCR products were further double size-selected to remove residual large gDNA fragments as described above. Purified PCR products were used in barcoding PCRs to add Illumina adapters and indices for pooled sequencing. A detailed protocol can be found on Addgene (https://www.addgene.org/pooled-library/human-transporters/).
 
Library strategy OTHER
Library source other
Library selection other
Instrument model Illumina HiSeq 4000
 
Data processing read trimming (Galaxy Version 4.8+galaxy1): options: g- GTGGAAAGGACGTGTCACCG…GTTTAAGAGCTA --overlap 10 --discard_untrimmed
gRNA mapping: MAGeCK count (Galaxy Version 0.5.9.2.4).
Assembly: GRCh38.p13
Supplementary files format and content: tab-delimited raw gRNA count table
Library strategy: Amplicon-Seq
 
Submission date Sep 20, 2024
Last update date Sep 25, 2024
Contact name Giulio Superti-Furga
Organization name CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
Street address Lazarettgasse 14
City Vienna
ZIP/Postal code 1090
Country Austria
 
Platform ID GPL27787
Series (1)
GSE277685 Identification of essential transporter genes in the colorectal carcinoma cell model HCT116 by focused CRISPR/Cas9 KO screens.
Relations
BioSample SAMN43846706
SRA SRX26142879

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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