2024-04-29
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114591046
External Id
CD45+
FACS
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA, UK University of Cambridge, Department of Haematology, Cambridge, CB2 0AW, UK Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute, Cambridge, CB2 0AW, UK
INSDC center name
2023-11-07T18:28:34Z
INSDC last update
public
INSDC status
ERS16703070
SRA accession
E-MTAB-13526_2:P6_T1
Submitter Id
69
year
age
ArrayExpress
broker name
not collected
collection date
human
common name
Protocols: Tissues were kept in cold complete RPMI medium (RPMI [Invitrogen] supplemented with 10% FBS [Sigma Millipore], 2mM L-Glutamine [Life Technologies] and 100U/ml Penicillin-Streptomycin [Thermofisher]) until dissection, which was performed on the same day of collection. Single-cell suspensions were generated as previously described25. Briefly, tissues were placed into a petri dish and cutted into small pieces of 2-4 mm and transferred into a 1.5ml tube containing the digestion mix (complete RPMI media supplemented with  1 mg ml−1 collagenase IV and 0.1 mg ml−1 DNase I) and minced using surgical scissors. Minced tissues were incubated for 45 minutes at 37℃ and vortexed every 15 minutes. Digested tissues were passed through a 100 μm strain into a falcon tube prefilled with cold PBS. Cells were then centrifuged for 5 minutes at 300 g, 4°C and the pellet was resuspended into 1x RBC lysis buffer (eBioscience) for 2 minutes at room temperature, after which 20 ml of cold PBS were added to stop the lysis reaction. Cells were cryopreserved in 5% DMSO in KnockOut Serum Replacement (KOSR; GibcoTM) until further use. On the day of the experiment, cells were rapidly thawed at 37℃ and transferred to complete RPMI media. Live cell enrichment was performed using MACS Dead Cell Removal Kit (Miltenyi Biotec) following the manufacturer's instructions. Red blood cells were further depleted by negative selection using CD235a Microbeads (Miltenyi Biotec) and MACS LS columns (Miltenyi Biotec), following the manufacturer's instructions. For FACS sorting, ce​lls were stained with Zombie Aqua to exclude dead cells and the cocktail of antibodies for 30 minutes at ​4°C. Cells were centrifuged for 5 minutes at 300 g, 4°C, resuspended in 500 μl of 5% FBS in PBS and subsequently filtered into polypropylene FACS tubes. Immune cells were sorted as live, CD45+; MDSC were sorted as live, CD45+, Lineage- (Lin: CD3, CD56, CD19), CD11b+, CD33+;. e-MDSC were sorted as live, CD45+, Lin-, CD33+, HLA-DR-/low. Cells were sorted into a 1.5 ml tube, counted and submitted for 10x scRNA seq library preparation. Each cell suspension was submitted for 3’ single cell RNA sequencing using Single Cell G Chip Kit, chemistry v3.1 (10x Genomics Pleasanton, CA, USA), following the manufacturer’s instructions. Each cell suspension was submitted for 3’ single cell RNA sequencing using Single Cell G Chip Kit, chemistry v3.1 (10x Genomics Pleasanton, CA, USA), following the manufacturer’s instructions.
description
adult
developmental stage
lung adenocarcinoma
disease
not collected
geographic location (country and/or sea)
Patient 6
individual
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
lung
organism part
TB20.0090
original source name
tumor
sampling site
Homo sapiens
scientific_name
female
sex
P6_T1
title
T1cN0M0
tumor grading