|
| Status |
Public on Oct 09, 2023 |
| Title |
PlasmidPool-for-Cas13d-TwoVector |
| Sample type |
SRA |
| |
|
| Source name |
Plasmid pool
|
| Organism |
synthetic construct |
| Characteristics |
sample type: PlasmidPool
tissue: NA
cell line: NA
infection: NA
time: NA
|
| Treatment protocol |
Cas13b/d-expressing A375 cells were firstly generated by lentiviral infection of Cas13b/d (pLentiCas13b/d-NES-blast) and then amplified to a number around 3x10^7. Then, these cells were infected with Cas13b/d crRNA-expressing lentiviral CRISPR library with MOI ~0.3. Two days later, select the infected cells with puromycin (1 µg/mL) for three days to get rid of any non-infected cells before changing back to normal media. At least ~300x coverage of cells were collected as Day 5 sample and stored at -80°C for later genomic DNA isolation. The rest of cells were continually cultured until Day 33 before harvesting as the end point sample.
|
| Growth protocol |
A375 cells were regularly tested negative for mycoplasma contamination and maintained in DMEM (SEVEN Biotech) supplemented with 10% fetal bovine serum (ExCell) and 1% penicillin–streptomycin (Solarbio). Cells were passaged every 2-4 days to maintain exponential growth and were kept in a humidity-controlled incubator at 37°C with 5% CO2. Protein-coding gene and lncRNA-targeting plasmid libraries under lentiviral backbone were firstly transfected along with pCMV8.74 and pMD2.G packaging plasmids into 293FT cells using Lipofectamine™ 2000 Transfection Reagent to generate crRNA-expressing lentivirus. Harvest virus-containing media at 72 hours post-transfection, and spin down the media at 1000 g for 5 min to remove the floating cells and cell debris. Carefully collect the virus supernatant, aliquot and store them at -80°C for further use. Test the virus titer and MOI (multiplicity of infection) before proceeding to the screen.
|
| Extracted molecule |
other |
| Extraction protocol |
The regions encompassing the sgRNAs were firstly PCR-amplified for around 20-23 cycles . The second round of PCR were employed to attach the illumina adaptors and index for around 10-12 cycles. These PCR products were gel purified and pooled for high-throughput sequencing to identify sgRNA abundance on illumina PE150 sequencing platform (Novogene, China).
The pooled synthesized oligos (Synbio Technologies, China) were PCR amplified and then cloned into pLentiGuideDR-puro vector (for expressing Cas13b/d crRNA) via BsmBI site by Gibson Assembly. The ligated Gibson Assembly mix was transformed into self-prepared electrocompetent Stable E. coli cells by electro-transformation to reach the efficiency with at least 100X coverage representation of each clone in the designed library. The transformed bacteria were cultured directly in liquid LB medium for 16~20 hours at temperature 30°C to minimize the recombination events in E. coli. The library plasmids were then extracted with EndoFree Maxi Plasmid Kit (TIANGEN, Cat no. 4992194).
Ensembl gene and lncRNA annotations (version: GRCh38) are used to extract the sequences of corresponding genes or lncRNAs. For genes (or lncRNAs) with multiple transcripts, the transcripts that have the corresponding RefSeq ID are used. We first enumerate all possible guides (22bp for Cas13d and 30bp for Cas13b) that span the entire transcript, then remove guides that (1) map to more than one location in the human genome and transcriptome (allowing up to 1 mismatch) or (2) contains BsmBI digestion sites (“CGTCTC” or “GAGACG”). The remaining guides are randomly selected if they hit the greatest number of transcripts (“common” guides) within the same gene/lncRNA. If all the guides that hit the greatest number of transcripts are selected, the remaining guides that hit the second greatest number of transcripts will be randomly selected, and so on. For essential and non-essential genes, 35 guides are designed per gene. For other genes or lncRNAs, 15 guides are designed per gene or lncRNA.
|
| |
|
| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
Pcr product from plasmid pool
|
| Data processing |
Read count table generated by MAGeCK 0.5.9.4
MAGeCK count command was used to collect read count from the fastq file
Assembly: hg38
Supplementary files format and content: tab-delimited text files include normalized counts for each Sample
|
| |
|
| Submission date |
May 12, 2022 |
| Last update date |
Oct 09, 2023 |
| Contact name |
Teng Fei |
| E-mail(s) |
feiteng@mail.neu.edu.cn
|
| Phone |
86-24-83656103
|
| Organization name |
Northeastern University, China
|
| Department |
College of Life and Health Sciences
|
| Lab |
Teng Fei's lab
|
| Street address |
195 Chuangxin Rd. Life Sciences Building A426. Hunnan District.
|
| City |
Shenyang |
| State/province |
Liaoning |
| ZIP/Postal code |
110169 |
| Country |
China |
| |
|
| Platform ID |
GPL26697 |
| Series (2) |
| GSE202841 |
Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems [Screen] |
| GSE202899 |
Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems |
|
| Relations |
| BioSample |
SAMN28204234 |
| SRA |
SRX15241470 |
