title: PBMC2_Illumina
organism: Homo sapiens
description: Protocols: iPSC: iPSC - Cells were harvested using ReleSRTM (Stem Cell Tech) and dissociated into a single cell suspension. Cells were counted and assessed for viability with Trypan Blue using a Countess II automated counter (Thermo Fisher Scientific), then pooled at a concentration of 391-663 cells/µL (3.91x105 - 6.63x105 cells/mL). TMWC: TMWCs were plated in T75 flasks and transfected with a pooled single guide RNA (sgRNA) library lentivirus containing sgRNA for 128 targets, 10 of which were control genes. Cells were harvested 7 days after virus transduction and were FACS sorted for EGFP-positive and viable cells (propidium iodide-negative cells) before applying to the Chromium System (10x Genomics) single cell RNA-sequencing workflow. PBMC1 and PBMC2: Peripheral blood samples were collected in Vacutainer Cell Preparation Tubes containing sodium heparin and ficoll (BD Biosciences: 362753), and were processed according to the manufacturer's recommendations. Following separation, PBMCs were cryopreserved and stored. Samples were subsequently thawed, and each library contained a pool of PBMCs from 14 donors, with 40,000 cells loaded to achieve a targeted 20,000 cells per library. TMWCs were transfected with a CROP-seq (Addgene: 99248) guide RNA (gRNA) pool targeting 128 loci, with the guides targeted to be inserted in the 3' end of the gene and thus detectable from short-read sequence data. TMWCs were plated in T75 flasks and transfected with a pooled single guide RNA (sgRNA) library lentivirus containing sgRNA for 128 targets, 10 of which were control genes. Samples were loaded on to the 10x Genomics Chromium Controller (10x Genomics, USA) for partitioning and barcoding. cDNA were extracted from all samples using the Single Cell 3' Library and Gel Bead Kit (V2; 10x Genomics; PN-120237).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-05-19T04:04:11Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-05-09T18:39:33Z
attributes.tag: External Id
attributes.value: SAMEA6820316
attributes.tag: INSDC center name
attributes.value: Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research
attributes.tag: INSDC first public
attributes.value: 2020-05-19T04:04:11Z
attributes.tag: INSDC last update
attributes.value: 2020-05-09T18:39:33Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9024:PBMC2_Illumina
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: peripheral blood mononuclear cell
attributes.tag: common name
attributes.value: human
attributes.tag: sample name
attributes.value: E-MTAB-9024:PBMC2_Illumina
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sequencing platform
attributes.value: NovaSeq 6000