|Public on Dec 16, 2016
|~4,000 bulk-sorted mouse ex-vivo myeloid cells
|strain: transgenic Cas9-GFP C57BL/7
age: 6 to 8 weeks-old
tissue: hind leg bones
tissue: bone marrow
selection marker: GFP+BFP+CD11c+
grna in the infecting vector: Itgam-BFP x Cebpb-mCherry
|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.
|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.
|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))
|Well with cells infected with a mix of Itgam-gRNA BFP and Cebpb-gRNA mCherry lentiviruses
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.
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.
|Nov 24, 2016
|Last update date
|May 15, 2019
|Weizmann Institute of Science
|234 Herzl st.
|Dissecting immune circuits by linking CRISPR pooled screens with single cell RNA-seq [SET2]
|Dissecting immune circuits by linking CRISPR pooled screens with single cell RNA-seq