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Sample GSM4297136

Query DataSets for GSM4297136

Status

Public on Feb 06, 2020

Title

D18_DMSO_r1

Sample type

SRA

Source name

Day 18 DMSO replicate 1

Organism

Homo sapiens

Characteristics

cell line: THP1
cell type: monocytic cell line derived from an acute monocytic leukemia patient

Treatment protocol

2x10^8 THP1 AML cells were infected with lentiviral particles at a multiplicity of infection of 0.3 and selected with 3ug/ml puromycin for seven days. 6×10^7 cells (representing 500-fold coverage of the library) were then washed with PBS and frozen for subsequent genomic DNA (gDNA) isolation. This was the early time point (Day 0). The remaining cells were treated with either DMSO or 250nM OG-86 for 18 days (sufficient for ten population doublings). Cells were divided between two groups of ten T225 flasks; each flask was seeded at 2×10^5 cells/mL in a total volume of 120mL. Cells were split every three days and medium and compound replaced. At the end of the experiment cells were frozen at -80°C for subsequent genomic DNA isolation using a QIAamp Blood Maxi kit as per manufacturer’s instructions.

Growth protocol

Human THP1 cells were from DMSZ and were cultured under standard conditions (5% CO2, 37°C) in RPMI1640 supplemented with 10% FBS, 10U/mL penicillin, 10μg/mL streptomycin and 2mM L-glutamine. Culture densities were 5x10^4 - 5x10^5 for cells in liquid culture.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was isolated using a QIAamp Blood Maxi kit as per manufacturer’s instructions.
To verify uniform representation of sgRNAs a two-step PCR was performed in which PCR1 amplifies the lentiviral sequence containing the 20bp sgRNA cassette and PCR2 attaches Illumina sequencing adapters and barcodes. In PCR#1, 50ng of each half library was amplified using V2 adaptor primers (http://genome-engineering.org/gecko/) using Phusion High Fidelity master mix (Thermo). The thermocycling conditions were: 98C for 3min, 15 cycles of (98C for 20s, 60C for 20s, 72C for 30s) and 72C for 7min. For each half library there were two PCR#1 replicates. 5uL of each #PCR1 product was run in 1.5% TAE-agarose ethidium bromide gel to verify amplicon length (~300bp). For the PCR#2 reaction, which is carried out to add appropriate Illumina sequencing adapters to the PCR#1 product, 5uL of each PCR#1 product was used as template. Unique barcoded forward and reverse primer combination were used for each PCR#2 reaction as described. Thermocycling conditions and cycle numbers were the same as for PCR#1. 5uL of PCR#2 product was run on a 1.5% TAE-agarose ethidium bromide gel to verify amplicon length (~350bp). All PCR#2 products were then pooled and gel purified from 2% TAE-agarose ethidium bromide gel using a QiaQuick kit, as per manufacturer’s instructions . The pooled library was then quantified by Qubit fluorometric quantitation and sequenced with 10% PhiX using a HiSeq 2500 System (Rapid v3). The sgRNA cassettes recovered from lentivirally-infected cells were amplified using PCR#1 and PCR#2 reactions as described above, but with some modifications. To preserve full library complexity and representation during sequencing, 244μg genomic DNA (~300-fold coverage) was used in each PCR#1 reaction per sample. For each PCR#1, we used 5ug gDNA using V2 adaptor primers and Phusion High Fidelity master mix (Thermo Fisher). The thermocycling conditions were: 98C for 30s, 19 cycles of (98C for 1s, 62C for 5s, 72C for 35s), 72C for 1min. 50 PCR#1 reactions were carried out per sample to capture the full representation of the screen. All the PCR#1 reactions for each biological sample were pooled together and 10uL of pooled #PCR1 product was used as template for PCR#2 reactions using barcoded primers for Illumina sequencing. The thermocycling conditions were: 98C for 30s, 19 cycles of (98C for 1s, 70C for 5s, and 72C for 35s), 72C for 1 min. For each sample, we performed at least 15 PCR#2 reactions (1 per 10K constructs). We included PCR#2 technical replicates for every sample (barcoded differently) to avoid duplicate sequences due to PCR over-amplification of constructs during sequencing. PCR#2 products of each technical replicate were pooled together and gel purified. The gel purified products were quantified by Qubit fluorometric quantitation (Thermo Fisher). Pooled PCR#2 products for each biological sample were then normalized before combining uniquely barcoded separate PCR replicates for all biological samples. The amount of amplifiable DNA was assessed by real time PCR using P5 and P7 Illumina primers. High throughput sequencing was performed with 15% PhiX and a HiSeq 2500 system.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

sgRNA_read_counts.txt

Data processing

Library strategy: CRISPR screen
The original FASTQ file was demultiplexed according to the 10 samples barcodes and the adaptors were trimmed using cutadapt (version 1.7.1). Then, the sgRNA guide sequence (20-mer) was aligned to the GeCKO library index (Human GeCKOv2 half libraries A and B) using bowtie by allowing up to one mismatch.
Supplementary_files_format_and_content: read counts

Submission date

Feb 05, 2020

Last update date

Feb 06, 2020

Contact name

Tim C Somervaille

E-mail(s)

tim.somervaille@cruk.manchester.ac.uk

Organization name

Cancer Research UK

Department

Manchester Institute