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| Status |
Public on Jun 19, 2018 |
| Title |
AB2597 |
| Sample type |
SRA |
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| Source name |
Lin- cKit+
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 Cas9-GFP
organ: bone marrow
selection marker: Lin- cKit+
treatment: Cas9-GFP LSK cells infected with lentiviral guide RNAs and transplanted into irradiated mice
lentiviral grna: Batf3_1, Batf3_2, Cebpa_1, Csf1r_1, Csf1r_2, CTL_LacZ, CTRL_LacOp, CTRL_Oris, Fcgr3_1, Fcgr3_2, Flt3_2, Irf8_C, Klf4_1, Klf4_2, Mta3_1, Mta3_2, Mta3_3, Satb1_3, Satb1_4, Spib_2, Tcf4_1, Tcf4_2, Tcf4_3
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| Treatment protocol |
Cas9-GFP LSK cells infected with BFP+ lentiviral guide RNAs and transplanted into irradiated mice. After 9-11 days, mice were sacrified, and GFP+ BFP+ cells were analyzed by CRISP-seq
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| Growth protocol |
6 to 8 weeks-old mice housed at the Weizmann Institute animal facility
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Each mouse was euthanized and bone marrow harvested by crushing of the femur, tibia and ilia. Cell suspensions were stained with FACS antibodies and single cells sorted into capture plates (Jaitin et al, Science 2014)
Antibodies used: Lin = Ter119 (TER-119), CD11b (itgam), Gr-1 (RB8/8C5), CD3 (17A2), CD4 (GK1.5), CD8a (53-6.7), B220 (RA3/6B2); and cKit (2B8), Sca1 (D7), CD34 (RAM34), Flt3 (A2F10), FcgR (93), CD150 (TC15-12F12.2), CD24 (M1/69), CD11c (N418), I-A/I-E (M5/114), CD19 (1D3), NK1.1 (PK136), CD71 (R17217), SiglecH(551), CD22 (OX-97)
3' end mRNA libraries were prepared for sequencing using CRISP-seq (Jaitin et al, Cell 2016)
DNA sequencing of guide RNA targeted loci for several CRISPR pools. Libraries were generated using primer flanking the target region for bulk population of cells, and sequenced.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
crispseq_count.txt
polyA+ lentiviral barcode
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| 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.
Note: Second read contained only cell and molecule barcodes. This information was appended to the fastq entry header
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 file that includes transcribed lentiviral barcodes, assigned to samples, single cells and infected gRNA
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| Submission date |
Apr 22, 2018 |
| Last update date |
Jun 19, 2018 |
| Contact name |
Ido Amit |
| E-mail(s) |
ido.amit@weizmann.ac.il
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| Phone |
972-8-9343338
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| Organization name |
Weizmann Institute of Science
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| Department |
Immunology
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| Street address |
234 Herzl st.
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| City |
Rehovot |
| ZIP/Postal code |
760001 |
| Country |
Israel |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE113494 |
Transcriptional plasticity, priming and commitment in hematopoietic lineages [CRISP-seq] |
| GSE113495 |
Transcriptional plasticity, priming and commitment in hematopoietic lineages |
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| Relations |
| BioSample |
SAMN08968552 |
| SRA |
SRX3984825 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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