GSM9339042: GE_Screen_Tcell; Mus musculus; OTHER
After 3-week ex vivo expansion of c-Kit+ bone marrow HSPCs, ~50 million cells were transduced overnight with lentivirus generated from one of 10 Bassik mouse CRISPR knockout sub-libraries40 (a kind gift from the Bassik lab; Addgene 1000000121-1000000130). Each sub-library contained ~20,000 elements. Lentivirus was generated using helper plasmids pCMV-VSV-G-RSV-Rev and pMDLg/pRRE. To reduce multiple integrations per cell, we used a transduction efficiency of 20-30%, to achieve 500-750x coverage (10-15 million transduced HSPCs per sub-library). Two days later, transduced HSPCs were selected for using puromycin for 72 hours. HSPCs were recovered and expanded in culture for another 9 days. For ex vivo timepoints, 20 million HSPCs were harvested for DNA extraction. At day-14 post-transduction, HSPCs were transplanted into lethally-irradiated (10Gy) C57BL/6-CD45.1 recipients (1-2 million HSPCs per recipient; >10 million per sub-library across 6-8 mice). A total of 65 mice were transplanted for the genome-wide screen, while 24 mice were transplanted for the Gene Expression-focused screen. After 10-12-weeks, whole bone marrow and spleen were harvested. From the whole bone marrow, c-Kit+Sca-1+Lineage- or c-Kit+Lineage- cells were isolated by FACS. The remaining bone marrow and spleen cells were pooled and CD45R+ B cells, CD4/CD8+ T cells, Mac1/Gr1+ myeloid cells, and Ter119+ erythroid cells were isolated via MACS columns. Genomic DNA was extracted for all screen populations separately according to the protocol included with QIAGEN Blood Mini or Midi Kit. Using known universal sequences present in the lentivirally incorporated DNA (oMCB 1562 and oMCB 1563), sgRNA sequences were amplified and prepared for sequencing by two sequential polymerase chain PCR reactions as previously described. Products were sequenced using an Illumina Nextseq to monitor library composition (20–40 million reads per sample).