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| Status |
Public on Jun 22, 2020 |
| Title |
CAL27_vivo_T1_Rep10 |
| Sample type |
SRA |
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| Source name |
CAL27
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| Organism |
Homo sapiens |
| Characteristics |
cell line: CAL27
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| Growth protocol |
SCC-13, CAL27, and A-431 cells were transduced with pLEX_Cas9 (Addgene: 117987) lentivirus and selected with blasticidin HCl for three to four days to establish stably expressing Cas9 lines. Cas9 expression was confirmed by anti-FLAG (F3165, Millipore Sigma) western blotting. Pooled guide lentivirus was titrated in each of the Cas9 expressing cell lines to achieve an MOI of 0.3. Following selection with puromycin for two to three days, three million cells were frozen at -80°C for the initial screen timepoint. Cells were cultured for three weeks for the in vitro CRISPR screen. For the in vivo CRISPR screen, two to four million cells were mixed with Matrigel (Corning) and injected into the rear flanks of SHO mice (for A431 and CAL27 lines) or NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (Jackson Laboratory) for SCC-13 cells. Resulting tumors were dissected after approximately two months. Tumors were minced and homogenized in an MP Biomedicals Benchtop Homogenizer (MP Biomedicals, Burlingame, CA) using MP Biomedicals lysing matrix A tubes.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from cells/tumors using the DNeasy Blood and Tissue Kit (Qiagen).
Two rounds of PCR were used to generate CRISPR screen sequencing libraries. To maintain guide library complexity, 36 100uL PCR 1 reactions were set up, each containing 500ng of genomic DNA, PrimeSTAR Max DNA Polymerase, and primers 3 and 4. PCR 1 reactions were heated at 98°C for 10 seconds, 56°C for 15 seconds, and 72°C for 15 seconds for a total of 5 cycles. Reactions were concentrated using the DNA Clean and Concentrator-100 kit (Zymo, Irvine, CA). PCR 1 primers were then removed using 1.1X AMPure XP beads (Beckman Coulter Life Sciences).
A single 100mL PCR 2 reaction was performed containing PrimeSTAR Max DNA Polymerase (Takara), the product from PCR 1, primers containing Illumina indexes and adapter sequences, and SYBR green (Thermofisher Scientific). The same cycling conditions used in PCR 1 were followed in PCR 2. The reaction was monitored on a Stratagene MX3005P quantitative PCR (qPCR) machine and stopped in the linear phase of amplification, typically after 20-25 cycles. The PCR 2 product was gel purified on a 2% agarose gel. The resulting library was analyzed on a Bioanalyzer and quantitated by qPCR with the KAPA Library Quantification Kit (Roche) prior to library mixing and deep sequencing on an Illumina MiSeq.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
sgRNA library
CAL27_vivo_gene_logFC.txt
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| Data processing |
Library strategy: CRISPR screen
Sequencing reads were first clipped using fastx clipper (from the FASTX-Toolkit package) for the sequence TCTTGTGGAAAGGACGAAACACCG. PCR duplicates were collapsed using the nine degenerate nucleotides added in PCR 1. Reads were then trimmed to the spacer sequence using fastx trimmer and mapped to the guide library using bowtie2. A read counts table was generated and used for downstream analysis.
The resulting matrix of read counts for each sgRNA (rows) and each sample (column) was then depth normalized so that each sample summed to ten million reads. These counts were log2-normalized, and the values for the time point 0 for each sgRNA were subtracted from time point 1 (three weeks of culture for in vitro and 8-10 weeks of xenograft growth for in vivo). Then for each sample, the median value of non-targeting sgRNAs was subtracted from all gene-targeting sgRNAs. These values are presented as “logFC” in the processed data tables.
Genome_build: NA
Supplementary_files_format_and_content: Processed data files contain the mean log2 fold-change of sgRNAs for each gene between the T1 time point (post-selection in vitro or xenograft tumor growth in vivo) and T0 time point (pre-selection). Each column of data represents a replicate in ascending order (Rep1, Rep2, Rep3, etc).
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| Submission date |
Jan 24, 2020 |
| Last update date |
May 02, 2022 |
| Contact name |
Andrew Ji |
| E-mail(s) |
andrew.ji@mssm.edu
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| Organization name |
Icahn School of Medicine at Mount Sinai
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| Street address |
1428 Madison Ave.
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL15520 |
| Series (2) |
| GSE144238 |
Single Cell and Spatial Analysis of Human Squamous Cell Carcinoma [CRISPR screen] |
| GSE144240 |
Single Cell and Spatial Analysis of Human Squamous Cell Carcinoma |
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| Relations |
| BioSample |
SAMN13919473 |
| SRA |
SRX7628682 |
| 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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