organism: Mus musculus
description: Protocols: Single cells RNA-Seq of CD45+ cells from lung tissues CD45+ cell isolation from lung tissues C57Bl/6J mice were injected with KP1.9 shCtrl or shPSME4 cells (0.25x10^6 cells, i.v.). After 21 days, lungs were perfused manually with 3 mL cold PBS w/o Mg/Ca through the right ventricle of the heart and harvested (4x shCtrl, 4x shPSME4) into cold PBS. The lung tissue was cut into pieces and processed using a GentleMACS Octo Dissociator (program 37C_m_LDK_1, Miltenyi Biotec) with the mouse lung dissociation kit (130-095-927, Miltenyi Biotec) according to the manufacturer's protocol. After the run, cells were strained through a 70 um mesh and washed with FACS buffer (0.5% BSA, 2 mM EDTA in PBS without Mg/Ca). Red blood cells were lysed with ACK buffer for 4 minutes, the reaction stopped by addition of cold FACS buffer. Finally, cells were resuspended in FACS buffer with FC block (anti-CD16/CD32, 1:500, Biolegend 101330) and incubated for 5 minutes. Lung cells from shCtrl or shPSME4 were stained with four differently labeled CD45 antibodies (APC, PE/Cy7, FITC, violetFluor 450) for 30 minutes at 4C. Cells were washed with FACS buffer twice, and the shCtrl or shPSME4 samples unified for parallel sorting of the four lung populations. Propidium iodide was added fresh (1 ug/mL) to exclude dead cells, and cells were sorted on BD FACSAria III and BD SORP FACSAria II sorters running on BD FACSDiva software (V8.0.1) using a 85 um nozzle. Finally, cells were washed twice (PBS w/o Mg/Ca, 0.04% BSA) and counted on a Neubauer chamber with trypan blue, and three samples from each condition were chosen (high cell count and viability >90%) for further processing. according to 10x Single cell RNA-seq using Chromium 10x genomics platform Single cell RNA-seq libraries were prepared at the Crown Genomics institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science. Cells were counted and diluted to a final concentration of approximately 1000 cells/uL in PBS supplemented with 0.04% BSA. Cellular suspension was loaded onto Next GEM Chip K targeting 6000 cells and then ran on a Chromium Controller instrument to generate GEM emulsion (10x Genomics). Single-cell gene expression libraries as well as single cell V(D)J libraries were generated according to the manufacturer's protocol using the Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) workflow. Final libraries were quantified using NEBNext Library Quant Kit for Illumina (NEB) and high sensitivity D1000 TapeStation (Agilent). Libraries were pooled according to targeted cell number, aiming for at least 5,000 reads per cell for V(D)J libraries and 20,000 reads per cell for gene expression libraries. Pooled libraries were sequenced on a NovaSeq 6000 instrument using an SP 200 cycles reagent kit (Illumina).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA14433908
attributes.tag: INSDC center alias
attributes.value: WIS
attributes.tag: INSDC center name
attributes.value: Weizmann Institute of Science
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:58:20Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:58:20Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10745:KP1.9 shPSME4 2
attributes.tag: age
attributes.value: 10
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: leukocyte
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: KP1.9 shPSME4
attributes.tag: immunophenotype
attributes.value: CD45+
attributes.tag: individual
attributes.value: 5
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: lung
attributes.tag: sample name
attributes.value: E-MTAB-10745:KP1.9 shPSME4 2
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL/6