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Sample GSM4284922

Query DataSets for GSM4284922

Status

Public on Jan 28, 2020

Title

HAP1 + LbCas12a + Optimization Library

Sample type

SRA

Source name

dual-guide amplicon

Organism

Homo sapiens

Characteristics

cell line: HAP1
cas protein: LbCas12a
crispr library: CHyMErA Human Optimization Libary
treatment: non-treated
library file: human_optimization_libV7_guides.fasta
annotation file: human_optimization_libV7_annot.txt

Treatment protocol

non-treated; 2.5 mM thymidine; 6 µM 6-thioguanine; Torin1 treatment was performed at a concentration that causes a 60% reduction in cell growth (i.e. IC60).

Growth protocol

Human HAP1 cells were maintained in low glucose (10 mM), low glutamine (1 mM) DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin. Human hTERT-RPE1 cells were maintained in DMEM with high glucose and pyruvate supplemented with 10% FBS and 1% Penicillin/Streptomycin. CGR8 mouse embryonic stem cells (mESC) were grown in gelatin-coated plates in GMEM supplemented with 100 μM β-mercaptoethanol, 0.1 mM nonessential amino acids, 2 mM sodium pyruvate, 2 mM L-glutamine, 5,000 units/mL penicillin/streptomycin, 1,000 units/mL recombinant mouse LIF and 15% ES fetal calf serum. Mouse neuroblastoma Neuro-2A (N2A) cells were grown in DMEM with high glucose supplemented with 10% FBS, sodium pyruvate, non-essential amino acids, and penicillin/streptomycin.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega).
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Sequencing libraries were prepared in two PCR steps, the first to enrich guide-RNA regions from the genome, and the second to amplify guide-RNA and attach Illumina TruSeq adapters with i5 and i7 indices.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Data processing

FASTQ files were pre-processed to extract Cas9 or Cas12a guide sequence using a bespoke Perl script.
The Cas12a guide was extracted from read1 files as the 23 bases preceeding 'ATCTAC', while Cas9 guide sequence was extracted from read2 files as the 20 bases proceeding 'GTTT'.
Trimmed FASTQ files were then aligned to the appropriate FASTA file using bowtie v0.12.1 using the following command line parameters: -p 16 -v 3 -l 18 --chunkmbs 256 -t <library_file> --un unmapped.fastq --al mapped.fastq -1 <trimmed_read1.fastq> -2 <trimmed_read2.fastq> aligned.sam
The number of reads aligning to each dual-guide sequence was enumerated for each aligned .sam file, and alignment statistics were recorded. Dual-guide IDs and counts were written to individual count files.
All individual count files were merged along with library annotations in R.

Submission date

Jan 27, 2020

Last update date

Feb 01, 2020

Contact name

Jason Moffat

E-mail(s)

j.moffat@utoronto.ca, jason.moffat@sickkids.ca

Phone

4379965938

Organization name

University of Toronto