|
| Status |
Public on Jul 26, 2019 |
| Title |
CHO_B |
| Sample type |
SRA |
| |
|
| Source name |
Starting library
|
| Organism |
synthetic construct |
| Characteristics |
cell type: Starting library
treatment: Untreated
selection: None
round of selection: 1
|
| Treatment protocol |
Cells were treated with histidinol to inhibit histidinyl-tRNA synthetase at 0.5 mM for 20 hours before collection and replating or sorting for dull cells and then replating. After recovery, some cells were expanded and aliquots saved for DNA prep or an additional round of sorting.
|
| Growth protocol |
Cells with a stable ISR reporter (CHOP::GFP) and stable Cas9 expression were infected with lentivirus prepared from pooled sgRNA expression library plasmid and helpers by transfection into HEK293T cells. After selection of the infected cells with puromycin for two weeks the cells were left untreated or treated and then sorted as indicated.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA containing the integrated sgRNA viral expression vectors was prepared using the DNAzol method.
Two round of PCR were used to produce barcoded amplicons that were pooled for sequencing. The first round of amplicon used vector directed primers flanking the 20bp sgRNA and the second round added barcodes to the individual samples.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiniSeq |
| |
|
| Description |
CLB_GCN_O1
Starting library_plasmid
processed data file: CHO_library_B_GCN Screen_counts.xls
|
| Data processing |
Library strategy: Amplicon sequencing
Compressed fastq files containing base calls were produced by the sequencing facility.
The MAGECK program (PMID: 25476604 PMCID: PMC4290824) was used for trimming and mapping the reads to the guide library (see supplementary file 'CHO_library_B_GCN Screen_counts.xls' columns A-F for library).
The raw guide counts were adjusted to total counts per sample to equalize between samples.
Genome_build: The genome used to build the guide list was GCF_000223135.1 (CriGri_1.0). Please see the supplementary file 'CHO_library_B_GCN Screen_counts.xls' columns A-F for the sg guide sequences and genes targeted.
Supplementary_files_format_and_content: *.txt: Tab-delimited text files, column A Guide unique ID, column B normalized guide counts.
|
| |
|
| Submission date |
Jul 25, 2019 |
| Last update date |
Jul 27, 2019 |
| Contact name |
Heather P Harding |
| E-mail(s) |
hph23@cam.ac.uk
|
| Organization name |
University of Cambridge
|
| Department |
CIMR Clinical Biochemistry
|
| Street address |
Hills Road
|
| City |
Cambridge |
| State/province |
Cambridge |
| ZIP/Postal code |
CB2 0XY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL26967 |
| Series (1) |
|
| Relations |
| BioSample |
SAMN12368409 |
| SRA |
SRX6596189 |
