Multiplexed_Library_1_Cplex
Homo sapiens
Protocols: PBMC from three healthy donors (Belgian Red Cross) were used for that experiment. Treg were isolated through immuno-magnetic selection and cultured for 24 hours under two conditions: Treg with IL-2 at 10 ng/mL alone or Treg with IL-2 at 10 ng/mL and TNF-α at 100 ng/mL. Cell concentration was 80,000 cells per 200 μL in a 96-well U bottom plate. After 24 hours, cells were collected and washed with 1 mL of PBS. The 10x Genomics 3' CellPlex Kit (10x Genomics B.V., Leiden, the Netherlands) was used to perform cell multiplexing: our six samples were labelled individually with cell multiplexing oligonucleotides. This allowed us to pool samples and read them together. Treg were isolated as previously described and cultured for 24 hours under two conditions: Treg with IL-2 at 10 ng/mL alone or Treg with IL-2 at 10 ng/mL and TNF-α at 100 ng/mL. 1) Cell multiplexing  As described in the kit protocol, 1 mL of PBS with BSA 0.04% (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were added to each sample (approximately 1 million cells per sample) which were then centrifuged at 400g for 5 minutes at RT. The supernatant was removed and cells were resuspended in 100 μL of Cell Multiplexing Oligo (one oligonucleotide per sample, 3' CellPlex Kit 502 Set A, 10x Genomics B.V.) by gently pipetting 10-15 times. Cells were incubated at RT for 5 minutes and mixed by gently pipetting with 1.9 mL of Wash and Resuspension buffer (composed of PBS and BSA 1%). Cells were centrifuged at 400g for 5 minutes at 4°C, the supernatant was removed, and two additional washes were made with 2 mL of the Wash and Resuspension Buffer at 400g for 5 minutes at 4°C (total of three washes). The supernatant was removed and cells were resuspended in the Wash and Resuspension Buffer to be counted (volume according to protocol) and assessed for viability using a Countness™ 2 Automated Cell Counter (Thermo Fischer Scientific). Next, labeled cells were pooled and the number of cells in the final mixture was counted.  2) Library preparation A total of 20,000 equimolarly pooled, CellPlex-labeled cells were processed using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 with Cell Multiplexing (10x Genomics, protocol CG000390) on a Chromium iX instrument with Chip G (10x Genomics).   3) Single-cell Gene Expression Matrices (GEMs) Generation GEMs were generated by combining barcoded Single Cell 3ʹ v3.1 Gel Beads, a Master Mix, and CellPlex-labeled cells with Partitioning Oil on a Chromium Chip G (10x Genomics). Reverse transcription was performed using a Veriti™ Thermal Cycler (Thermo Fisher Scientific) with the following conditions: 53°C for 45 minutes, 85°C for 5 minutes, and holding at 4°C.  3) cDNA Amplification and Purification  Following emulsion breakage, pooled fractions were purified using DynaBeads MyOne Silane Beads (10x Genomics). cDNA and CellPlex barcodes were amplified via PCR with a 12-cycle reaction module: 98°C for 3 minutes, 98°C for 15 seconds, 63°C for 20 seconds and 72°C for 1 minute, followed by a final extension at 72°C for 1 minute. Feature cDNA Primer 3 was used to amplify both cDNA and CellPlex (primer included in the kit).  The cDNA reaction was purified using magnetic SPRI Beads (Beckman Coulter, Indiana, United States). The supernatant, containing barcoded CellPlex amplicons, was separated and purified again for CellPlex library preparation. 1) Library Construction for 3' Gene Expression  For 3ʹ Gene Expression libraries, amplified cDNA was fragmented, end-repaired, A-tailed and ligated to sequencing adapters. Libraries were then amplified by PCR (indexing PCR) using Dual Index TT primers plate (10x Genomics).  2) CellPlex Library Construction  Purified CellPlex amplicons were processed in an indexing PCR using Feature SI Primers 2 (included in the kit) with dual single index primers (Dual Index Kit NN Set A, 10x Genomics).
ENA first public
2025-11-20
INSDC center name
1) Hematology Research Unit, Groupe Interdisciplinaire de Génoprotéomique Appliquée, (GIGA)-I³, University of Liège, Liège, Belgium 2) Department of Medicine, Division of Hematology, CHU of Liège, Liège, Belgium.
INSDC status
public
Submitter Id
E-MTAB-15881:Multiplexed_Library_1_Cplex
broker name
ArrayExpress
cell line
PBMC
cell type
regulatory T cell
collection date
not collected
common name
human
developmental stage
adult
disease
normal
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
blood
protocol
Cplex
sample name
E-MTAB-15881:Multiplexed_Library_1_Cplex
scientific_name
Homo sapiens