|
Status |
Public on Dec 16, 2016 |
Title |
AB1564 |
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: developmental genes mix (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.
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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.
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|
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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, Spi1, Irf8 and control gRNA lentiviruses (all with the BFP selection marker)
|
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
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|
|
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 |
SAMN06053594 |
SRA |
SRX2373861 |