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Sample GSM8329974

Query DataSets for GSM8329974

Status

Public on Jun 19, 2024

Title

Cas12a-SLCxSLC_sublib2_input_rep2

Sample type

SRA

Source name

Plasmid

Organism

synthetic construct

Characteristics

tissue: Plasmid
cell line: -
cell type: -
genotype: -
treatment: --

Treatment protocol

Cas9 pools were cultivated in standard media (glu) or in hypoxia (hyp) conditions (1% oxygen), if indicated. Cas12a pools were cultivated in standard media (glu), under hypoxia (hyp), in presence of 50 mM antimycin (ant) or in low glucose media with galactose (gal) replacement (week 1-3: 20% glucose/80% galactose, week 3-4: 100% galactose, week 4-5: 10% glucose/90% galactose), if indicated. Treatments were started after one (Cas12a samples) or two (Cas9 samples) weeks after transduction. Cells from Cas12-SLCxSLC and Cas9-SLCxSLC samples were cultured in the presence of 100 U/ml Nystatin (Merck, #N3503-5MU) to prevent fungal contaminations.

Growth protocol

For standard culture conditions, HCT116 cells were grown in RPMI (R8758 Sigma) supplemented with 10% Fetal Bovine Serum (10270-106, Lot 42F8381K, Gibco) and Penicillin-Streptomycin (15140-122, Gibco)

Extracted molecule

other

Extraction protocol

For the Cas12a-Metal-SLCxSLC, Cas12a- and Cas9-SLCxEnzymes, and Cas12a- and Cas9-SLCxSLC, gDNA was purified from 2x10e7 (QIAamp DNA Mini Kit), 1.7x10e7 (QIAGEN Genomic-tip 100/G) or 2.2x10e8 cells (QIAGEN Genomic-tip 500/G), respectively. gDNA from Cas12a- and Cas9-SLCxSLC screens was digested with restriction enzymes to improve PCR amplification of sgRNA regions. Up to 1.5 mg gDNA was digested with 200 U PacI (Cas12a-SLCxSLC) or 100 U PacI and 100 U XbaI (Cas9-SLCxSLC) (all NEB) for 48 hours.
sgRNA sequences from Cas12a library gDNA samples were amplified with barcoded FW and RV primer that introduce Illumina adapter and read1 sequencing primer binding sites. Up to 1.2 mg gDNA was used as template in 12 ml volume (divided in 96 x 125 µl PCRs) in the following program: 98°C for 30 sec.; 5 cycles: 98°C for 10 sec., 61°C for 30 sec, 72°C for 30 sec; 19 cycles: 98°C for 10 sec., 72°C for 1 min.; 72°C for 2 min. For the samples of the metal transporter and SLC family-wide sub-screen 1, Q5 Polymerase (NEB) was used, SLC vs SLC sub-screen 2-4 and SLC vs metabolic enzymes samples were processed with self-purified Pfu-Sso7d polymerase and Phusion HF buffer (NEB). Primer sequences are shown in Supplementary Table SX. PCR products we double size selected with DNA purification bads and multiplexed for Illumina sequencing. sgRNA sequences from Cas9 library gDNA samples were first amplified with staggered FW and RV primer that contain partial Illumina adapter sequences at the 5’ end using the same PCR program as above. Up to 1.2 mg gDNA was used as template (125 µg/125 µl volume PCR) and amplified with self-purified Pfu-Sso7d polymerase. An aliquot of the pooled amplified PCR products was double size selected with DNA purification bads and purified fragments were barcoded in a second PCRs (98°C for 30 sec; 6-8 cycles: 98°C for 10 sec., 61°C for 30 sec, 72°C for 30 sec; 72°C for 2 min). PCR products we double size selected with DNA purification bads and multiplexed for Illumina sequencing.

Library strategy

OTHER

Library source

other

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

Combinatorial Cas12a library

Data processing

read trimming (Cas9 libraries only): Read1 reads were trimmed using cutadapt (Galaxy Version 4.8+galaxy1) to remove up- and downstream flanking sequences. The two gRNAs from read1 and read2 were then joined using FASTQ Joiner (Galaxy Version 2.0.1.1+galaxy0) before mapping to the reference sequences.
read mapping: MAGeCK count (Galaxy Version 0.5.9.2.4).
Assembly: Human NCBI genome build 36
Supplementary files format and content: tab-delimited raw gRNA pair count tables
Library strategy: Amplicon-Seq

Submission date

Jun 14, 2024

Last update date

Jun 19, 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