title: TMWC_BGI
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. cDNA libraries were created in using the 10x Genomics Single Cell 3' Library and Gel Bead Kit (V2; 10x Genomics; PN-120237). For compatibility with BGI sequencers, the libraries underwent a conversion step via the MGIEasy Universal Library Conversion kit (App-A) (Part Number: 1000004155).
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: SAMEA6820317
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:TMWC_BGI
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: trabecular meshwork cell
attributes.tag: common name
attributes.value: human
attributes.tag: sample name
attributes.value: E-MTAB-9024:TMWC_BGI
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sequencing platform
attributes.value: MGISEQ-2000