|
| Status |
Public on Dec 16, 2016 |
| Title |
AB1594 |
| Sample type |
SRA |
| |
|
| Source name |
single cell-sorted mouse ex-vivo myeloid cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: transgenic Cas9-GFP C57BL/6
organ: bone marrow-derived
selection marker: GFP+BFP+CD11c+
treatment: cells stimulated with lypopolysacharide (LPS) for 4 hours
grna in the infecting vector: INF/AV mix1 (BFP)
|
| Treatment protocol |
Cells were infected on day 2 post plating with lentivirus vectors, each containing one gRNA and one fluorescent selection marker. On day 7, cells were challenged with LPS for 4 hours before harvest.
|
| Growth protocol |
Bone marrows were flushed from hind leg bones from euthanized 6 to 8 weeks-old mice housed at the Weizmann Institute animal facility. Cell suspensions were obtained, red blood cells lysed, and cells grown on 6-well petri dishes on media supplemented with GM-CSF.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were scrapped from the plate and stained with FACS markers antibodies as indicated above (selection marker).
3' end mRNA libraries were prepared for sequencing using CRISP-seq (Jaitin et al., Cell 2016), which includes a module of MARS-seq (Jaitin et al., Science 2014) and a module of UGI-seq.
single cell RNA-seq and UGI-seq for gene expression quantitation and gRNA identification, respectively.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Well with cells infected with a mix of Cebpb, Rela, Nfkb1, Nfkb2, Stat2, Irf9 and control gRNAs and BFP lentiviruses
|
| Data processing |
bcl2fastq/2.15.0.4
Sequences with RMT of low quality (defined as RMT with minimum Phred score of less than 27) were filtered out.
Pool-barcode and well-barcode-RMT were extracted from the first and second end of the read (respectively) and concatenated to the fastq header, delimited by a underscore i.e. POOL_BARCODE_WELL_BARCODE_RMT while "NNNNNN" was used as a place holders if plate barcode was not used.
Reads were separated by POOL_BARCODE_WELL_BARCODE header data, allowing 1 sequencing error. This process created a single fastq file for each source well.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include mRNA molecule count values for each Sample; single cell expression matrix
|
| |
|
| Submission date |
Nov 24, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ido Amit |
| E-mail(s) |
ido.amit@weizmann.ac.il
|
| Phone |
972-8-9343338
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Immunology
|
| Street address |
234 Herzl st.
|
| City |
Rehovot |
| ZIP/Postal code |
760001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE90486 |
Dissecting immune circuits by linking CRISPR pooled screens with single cell RNA-seq [SET1] |
| GSE90488 |
Dissecting immune circuits by linking CRISPR pooled screens with single cell RNA-seq |
|
| Relations |
| BioSample |
SAMN06053584 |
| SRA |
SRX2373871 |
