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Sample GSM7623685

Query DataSets for GSM7623685

Status

Public on Oct 01, 2023

Title

CSF1 stimulated Input sgRNAs

Sample type

SRA

Source name

Plasmid DNA used to make lentivirus

Organism

synthetic construct

Characteristics

strain: n/a

Treatment protocol

Primary Macrophages expressing Cas9-GFP were transduced with the Brie Library at low multiplicity of infection (MOI) (0.3) to achieve 50 fold representation of each sgRNA in the Brie Mouse library. After 48 h, transduced cells were selected with 5 ug/ml puromycin for 2 days and cultured another 8 before sorting. To identify genes regulating dextran uptake, cells were starved of CSF1 overnight in DMEM plus 10% FBS. Then, cells were exposed to 300 ug/ml 40 kDa fluorescent dextran for 15 minutes in the presence or absence of CSF1. Cells were then sorted based on their ability to internalize dextran using a BD FACs jazz flow cytometer. We sorted the top (high fluorescence) and bottom (low fluorescence) quintiles based on dextran fluorescence and sequenced both samples to idenfity sgRNAs enriched in each sample.

Growth protocol

BMDM were cultured in bone-marrow medium (BMM) containing Dulbecco’s Modified Eagle Medium (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate), 20% heat-inactivated fetal bovine serum and 30% L-cell supernatant (a source of colony-stimulating factor-1), 10,000 IU penicillin and 10 mg/mL streptomycin, and 5.7 mM 2-mercaptoethanol.

Extracted molecule

other

Extraction protocol

Genomic DNA was extracted from cells using the GeneJET genomic DNA extraction kit (#K0721 Thermofisher) and the sgRNA library was amplified by a single-step PCR protocol (Broad Institute) for NGS using barcoded primers with staggered sequences. PCR product from multiple PCR reactions was pooled and gel purified and quantified by Qubit Fluorometer and subjected to sequencing on Illumina Nextseq 500 with 75 reads.
Mouse Brie CRISPR knockout pooled library developed by David Root and John Doench was obtained from Addgene (Addgene #73633) was amplified in Stable 3 competent E. coli (Thermofisher) and subjected to next-generation-sequencing (NGS) to determine the distribution of sgRNAs in the library. To produce Lentivirus, 1.8 x 106 NIH 293T cells were seeded in six 10-cm plates and transfected the following day with 6 μg sgRNA pooled library in lentiGuide-Puro (Addgene# 73633), 6 μg psPAX2 plasmid, 1 μg pVSVG plasmid, and 0.024 mg polyethyleneimine (PEI). After 48 h, the lentivirus was harvested, centrifuged, and aliquoted into multiple 15 mL tubes and stored at -80o C or - 150o C. The functional titer of the lentivirus was determined by live-cell counting after puromycin selection.
The parameters for how the library sequenced - chip: 75 high fidelity read

Library strategy

OTHER

Library source

other

Library selection

other

Instrument model

Illumina NextSeq 500

Description

This sample represents the sgRNAs used to transduce cells with for the CSF1 stimulated dextran uptake screen
CSF1stim.count.txt

Data processing

20 base sgRNA reads were trimmed using Cutadapt 2.3, where 20bp sequence flanked by cgaaacaccg...gttttagagc was identifed from all fastq files. Cutadapt arguments= -g cgaaacaccg...gttttagagc, -o, --minimum-length 15, --discard-untrimmed, -e 0.2, --cores=8
Counts of sgRNA reads generated and mapped to Brie library sgRNAs using MAGeCK 0.5.9. arguments= count, -l BrieGuideLibwithNonTarg.csv, --sample-label, --fastq, -n, --unmapped-to-file, --pdf-report
We used the MAGeCK test command to identify enriched sgRNAs within the high and low populations. Arguments= test, -k, -c low, -t high, -n, --control-sgrna nontargets.txt --keep-tmp --pdf-reports
Assembly: Brie Library
Supplementary files format and content: .txt and .csv files contain the data files used to process the data. The BrieGuideLibwithNonTarg.csv file contains the Brie library file used by MAGeCK to map the trimmed reads back to the library. The readmapping step generated the "CSF1stim.count.txt" and "unstim.count.txt" files used for the MAGeCK test functions. The MAGeCK test step used one of the count tables and the "nontargets.txt" file to identify sgRNAs enriched in the low and high fluroescence populations. The MAGeCK test output includes the "CSF1_dexuptake_highlow.gene_summary.txt" and "unstim_dexuptake.gene_summary.txt" files. The MAGeCK out files were used to identify genes regulating dextran uptake.
Library strategy: CRISPR screen

Submission date

Jul 17, 2023

Last update date

Oct 01, 2023

Contact name

Natalie W Thiex

E-mail(s)

natalie.thiex@sdstate.edu

Phone

605-688-5874

Organization name

South Dakota State University