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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 12, 2018 |
| Title |
Shank2 splicing reporter, sorted cells with 2% lowest EGFP/mCherry, replicate 4 (T22) |
| Sample type |
SRA |
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| Source name |
Neuroblastoma cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: N2A
cell type: Neuroblastoma
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| Treatment protocol |
80 million N2A Flp-In cells expressing bichromatic splicing reporters were infected with the lentiviral Brie library (78,637 gRNAs) at an MOI ~0.3, in the presence of 8 μg/ml polybrene, such that every sgRNA was represented in approximately 300 cells. 24 hours after infection, the medium was replaced with fresh medium containing Puromycin (2.5 μg/ml) and cells were incubated for an additional 72 hours. After selection cells were split such that each sgRNA would be represented by an average of 300 cells in the population (i.e. 24 million cells) and passaged every three days. For cell sorting, the 24 million cells were seeded and the next day 1 μg/ml doxycycline was added to induce bichromatic reporter expression. 24 hours later the cells were harvested and resuspended in sorting buffer (Hanks Balanced Salt Solution, 25 mM HEPES pH 7.0, 1 mM EDTA, 1% Albumin) at a concentration of 5 million cells per ml. The cells were passed through a nylon mesh with a pore size of 40 μm to eliminate large aggregates. Filtered cells were sorted based on the relative EGFP:mCherry expression FACS. For each replicate of the lowest/highest 2% sorting scheme, 300,000-500,000 cells were collected. For the 30% sorting scheme, the same procedure was followed except that the 30% of cells with the highest or lowest EGFP:mCherry ratio were sorted. In addition, 24 million cells were also collected prior to sorting as a reference point for comparing enrichment in the sorted populations.
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| Growth protocol |
Mouse neuroblastoma (N2A) and 293T cells were grown in DMEM (high glucose; Sigma-Aldrich) supplemented with 10% FBS, sodium pyruvate, non-essential amino acids, and penicillin/streptomycin, at 37°C with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
cell engineering protocol: A Flp-in cassetted was integrated into the Rosa26 locus in N2a cells by first creating cells with an FRT site in that locus, followed by integration of a Flp-In construct from pcDNA5/FRT/TRE modified to include an rtTA3-compatible Tet Response Element (TRE). A bichromatic microexon splicing reporter based on a microexon from either murine Shank2 (ENSMUSE00001006087) or Mef2d (ENSMUSE00000673645) was then constructed by amplifying the microexons (extended by one nucleotide to cause a frame shift), flanking introns, upstream constitutive exon, and 20-50 nt of the downstream constitutive exon 5' of a fluorescence cassette consisting of EGFP and mCherry in different reading frames. This construct was cloned into a customized pcDNA5-based Gateway(R) compatible vector (Life Technologies) that contained 8x TET response elements followed by a miniCMV promoter, Kozak sequence, 3x Flag-tag, and attR sites, followed by integration in a clonal N2A Flp-In cell line.
Genomic DNA was extracted from the cell pellets of unsorted samples using the QIAamp Blood Maxi Kit (Qiagen) while gDNA from sorted populations was purified with the Midi Kit as per the manufacturer’s recommendations. Genomic DNA was precipitated using ethanol and sodium chloride, and resuspended in Buffer EB (10 mM Tris-HCl, pH 7.5).
gRNA inserts were amplified via PCR using primers harboring Illumina TruSeq adapters with i5 and i7 barcodes, following a two-step PCR approach. In the first step, a total of 50 μg of gDNA was subjected to PCR (25 cycles, temperature of annealing (Ta) = 65°C) for enrichment of sgRNA cassettes using NEBNext Ultra II Q5 polymerase and staggered primers annealing to the end of the U6 promoter and the beginning of the tracrRNA. For the 2% highest or lowest expressing sorted cell populations 10 μg of gDNA was used. Subsequently, the individual PCR reactions were pooled and 50 μl of the first step PCR product was loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1.5 hour at 100V. PCR products of 200-230 bp were selected and the excised gel fragments were purified using the Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen). 1/6th of the purified PCR products were subjected to 2nd step PCR (10 cycles, Ta= 62°C) using NEBNext Ultra II Q5 polymerase and primers harboring Illumina TruSeq adapters with i5 and i7 indices to generate barcoded sequencing libraries ready for Illumina sequencing (Table S8). The PCR amplicons were loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1 hour at 100V. The libraries were size-selected at 270 bp and 300 bp, and the excised gel fragments were purified using Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen). The purified libraries were analyzed for quality control, pooling and sequencing. Size confirmation analysis for each sample was performed on an Agilent Bioanalyzer dsDNA High Sensitivity chip. Amplicons were quantified using Kapa Universal qPCR Master Mix (Cat # KK4923, Roche) and Qubit dsDNA HS Assay Kit (Cat # Q32854, Thermo Fisher Scientific) and pooled at varied ratios by molarity after size-adjustment. To mitigate the effects of index hopping, pooling was performed just prior to sequencing. The final pool was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip and quantified using NEBNext Library Quant Kit for Illumina (Cat # E7630L, NEB).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_30
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| Data processing |
Library strategy: CRISPR screen
Base calls were processed with bcl2fastq2 (Illumina) version 2.18.
sgRNA sequences were extracted from raw reads based on matches to the tracrRNA and U6 promoter using custom Perl scripts, and perfect matches to expected sgRNAs were tallied.
To score sgRNAs that impact reporter fluorescence, raw counts from sorted and unsorted populations were analyzed with the count module of MAGeCK version 0.5.6 (Li et al., 2014) with default settings where 1,000 non-targeting control sgRNAs present in the Brie library were specified with parameter –control-sgrna.
Genome_build: Brie mouse sgRNA library (Addgene #73633)
Supplementary_files_format_and_content: Tab delimited files with counts per sgRNA sequence in each sample; MAGeCK.scores.xlsx is a speadsheet containing the results of MAGeCK analyses, i.e. the enrichment of genes in the sorted cell polulations.
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| Submission date |
Apr 02, 2018 |
| Last update date |
Oct 12, 2018 |
| Contact name |
Ulrich Braunschweig |
| Organization name |
University of Toronto
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| Department |
Donnelly Centre
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| Lab |
Benjamin J. Blencowe
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| Street address |
160 College Street
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE112599 |
Genome-wide CRISPR-Cas9 interrogation of splicing networks reveals a mechanism for recognition of autism-misregulated neuronal microexons [CRISPR screen] |
| GSE112601 |
Genome-wide CRISPR-Cas9 interrogation of splicing networks reveals a mechanism for recognition of autism-misregulated neuronal microexons |
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| Relations |
| BioSample |
SAMN08832659 |
| SRA |
SRX3873123 |
| 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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