|
| Status |
Public on Dec 16, 2016 |
| Title |
SP-Itgam_Cebpb.sca |
| Sample type |
SRA |
| |
|
| Source name |
~4,000 bulk-sorted mouse ex-vivo myeloid cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: transgenic Cas9-GFP C57BL/7
age: 6 to 8 weeks-old
tissue: hind leg bones
tissue: bone marrow
selection marker: GFP+BFP+CD11c+
treatment: unstimulated
grna in the infecting vector: Itgam-BFP x Cebpb-mCherry
|
| 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 |
genomic DNA |
| 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 (paired-end + i7 for sample index (7 bases))
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Well with cells infected with a mix of Itgam-gRNA BFP and Cebpb-gRNA mCherry lentiviruses
Sample 53
|
| 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: lists of gene counts; specific amplicons within genomic DNA;
Supplementary_files_format_and_content: CIGAR string as defined in SAM file sprcifications, showing count of uniq strings. This is used to estimate the editing percentage of the target loci.
|
| |
|
| 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 |
GPL16417 |
| Series (2) |
| GSE90487 |
Dissecting immune circuits by linking CRISPR pooled screens with single cell RNA-seq [SET2] |
| GSE90488 |
Dissecting immune circuits by linking CRISPR pooled screens with single cell RNA-seq |
|
| Relations |
| BioSample |
SAMN06053563 |
| SRA |
SRX2373900 |
