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GEO help: Mouse over screen elements for information. |
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Status |
Public on Jan 28, 2020 |
Title |
N2A + AsCas12a + mouse Optimization Library |
Sample type |
SRA |
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Source name |
dual-guide amplicon
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Organism |
Mus musculus |
Characteristics |
cell line: N2A cas protein: AsCas12a crispr library: CHyMErA Mouse Optimization Libary treatment: non-treated library file: mouse_optimization_libV3_guides.fasta annotation file: mouse_optimization_libV3_annot.txt
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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).
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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.
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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.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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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.
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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
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Phone |
4379965938
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Organization name |
University of Toronto
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Department |
Molecular Genetics
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Street address |
686 Bay Street, Room 13.9705
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City |
Toronto |
State/province |
ON |
ZIP/Postal code |
M5G0A4 |
Country |
Canada |
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Platform ID |
GPL24247 |
Series (1) |
GSE144281 |
Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform |
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Relations |
BioSample |
SAMN13927453 |
SRA |
SRX7635173 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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