community.elabeln.com/experiments.php?mode=edit&id=314
#314
Genetics & Genomics
Complete
scRNA-Seq: PBMC donor 7 / 10x 3' v3.1 (10k cells)
This template captures a single-cell RNA-Seq profiling run of human peripheral blood mononuclear cells (PBMCs) from a healthy donor (donor 7), generating an 8-cluster immune cell atlas using the 10x Genomics Chromium platform.
Goal: capture ~10,000 high-quality single-cell transcriptomes from donor PBMCs, identify the major immune cell types (T cells, NK cells, B cells, monocytes, dendritic cells, platelets) with cluster-marker validation, and establish a baseline reference for downstream perturbation studies.
Method overview: PBMCs isolated from 8 mL fresh whole blood by SepMate density gradient, washed in 1× PBS + 0.04% BSA, counted on Countess II to 1,200 cells/µL viable. Target 10,000 cells loaded onto Chromium Next GEM Chip G, paired with Single Cell 3′ v3.1 Gel Beads. GEM-X partitioning on Chromium Controller, RT in droplet, cleanup, cDNA amplification, library construction per 10x CG000204 user guide. Library QC on BioAnalyzer HSDNA + Qubit dsDNA HS. Pooled and sequenced on NovaSeq 6000 SP flow cell (R1 28 bp barcode + UMI, R2 90 bp cDNA insert). Demultiplexed BCL → FASTQ via bcl2fastq, aligned + counted via Cellranger count v8.0 against GRCh38. Loaded into Seurat v5 for QC filtering (200-7500 genes/cell, <15% mito), normalized with SCTransform, dimensionality reduction (PCA + UMAP), Leiden clustering at resolution 0.6, cell-type annotation via Azimuth + manual marker curation.
Outcome: 10,182 cells captured (target 10k), 9.0% doublet rate (acceptable for v3.1), 28,124 mean reads per cell, 1,850 median genes per cell, 1.2% mitochondrial fraction (low — indicates healthy cells), 92% sequencing saturation (well-saturated library). 8 cell-type clusters resolved spanning CD4 T (24%), CD8 T (18%), NK (12%), B (16%), classical Monocyte (15%), mDC (4%), pDC (2%), Platelet (9%).
Last saved: 34 seconds ago
12 fields loaded from scRNA-Seq 10x baseline (SOP v1.8)
Sample
Tissue
select
PBMC, whole blood, dissociated tumor, brain, lung, intestine, organoid
Donor
text
Donor 7 (healthy)
Dissociation
select
density gradient, mechanical, enzymatic (Collagenase + DNase)
Capture & Library
Chemistry
select
10x 3' v3.1, 10x 5' v2, 10x multiome, 10x fixed RNA, 10x multiplex
Target cells
number
10000
Captured cells
number
10182
Doublet rate
number
9.0%
Sequencing & QC
Mean reads/cell
number
28124
Median genes/cell
number
1850
Mito fraction
number
1.2%
Saturation
number
92%
Clusters
number
8
01
0:10
Receive 8 mL fresh whole blood from donor 7, transfer to SepMate tubes
02
0:40
PBMC isolation: SepMate density gradient, 1200×g × 10 min, wash 2× in 1× PBS + 0.04% BSA
03
0:08
Count viable cells on Countess II, resuspend to 1,200 cells/µL for ~16k cells loading
04
0:15
Prepare master mix + load Chromium Next GEM Chip G with target 10,000 cells per channel
05
0:12
Run Chromium Controller for GEM-X partitioning + droplet generation
06
4:00
Reverse transcription in droplets (53°C × 45 min), cleanup, cDNA amplification (12 cycles)
07
3:30
Library construction per 10x CG000204: fragmentation, adapter ligation, sample index PCR
08
0:30
Pool with 3 other libraries (equimolar), submit to seq core for NovaSeq SP flow cell
09
12:00
BCL→FASTQ via bcl2fastq, Cellranger count v8.0 alignment + UMI quantification (GRCh38)
10
4:00
Seurat v5 QC + clustering + Azimuth annotation, archive .rds + cluster marker tables
| A | B | C | D | E | |
|---|---|---|---|---|---|
| 1 | Cluster | Cell type | N cells | Marker genes | Notes |
| 2 | 0 | CD4 T cell | 2444 | CD3D, CD4, IL7R, CCR7 | Naive + memory mixed |
| 3 | 1 | CD8 T cell | 1833 | CD3D, CD8A, GZMA, GZMK | Includes effector + memory |
| 4 | 2 | NK cell | 1222 | GNLY, NKG7, KLRD1, NCAM1 | CD56-bright + CD56-dim mixed |
| 5 | 3 | B cell | 1629 | CD19, CD79A, MS4A1, CD37 | Naive B dominant |
| 6 | 4 | Classical Monocyte | 1527 | CD14, LYZ, S100A8, S100A9 | CD14++ CD16- |
| 7 | 5 | mDC | 407 | CD1C, FCER1A, HLA-DQB1 | Conventional DC2 |
| 8 | 6 | pDC | 204 | IL3RA, LILRA4, IRF7, GZMB | Plasmacytoid DC |
| 9 | 7 | Platelet | 916 | PPBP, GP9, ITGA2B, PF4 | Doublet-like, kept after manual review |
EXP-2026-0530
Donor 7 IRB consent + sample chain of custody
R. Tanaka
May 30
EXP-2026-0612
CD8 T trajectory analysis (Monocle3)
R. Tanaka
Jun 12
SOP-0438
scRNA-Seq 10x 3' v3.1 SOP v1.8
QA Team
Apr 19
CG000204
10x Chromium Next GEM Single Cell 3' v3.1 user guide
10x Genomics
Feb 7
PIPE-SC1
Seurat v5 analysis pipeline reference docs
Bioinformatics
Jan 22
Visibility
Tanaka LabR. Tanaka
Can write
R. TanakaDr. E. Kim
Use Ctrl + click to add text
{
"experiment": {
"id": 314,
"custom_id": "SCRNA-D7-2026-003",
"title": "scRNA-Seq: PBMC donor 7 / 10x 3' v3.1 (10k cells)",
"template": "Single-Cell RNA-Seq",
"template_version": "v3.2",
"category": "Genetics & Genomics",
"status": "Complete",
"started_on": "2026-07-15",
"tags": [
"scRNA-Seq",
"10x-Chromium",
"PBMC",
"Seurat",
"immune-profiling"
]
},
"main_text": "This template captures a single-cell RNA-Seq profiling run of human peripheral blood mononuclear cells (PBMCs) from a healthy donor (donor 7), generating an 8-cluster immune cell atlas using the 10x Genomics Chromium platform.\nGoal: capture ~10,000 high-quality single-cell transcriptomes from donor PBMCs, identify the major immune cell types (T cells, NK cells, B cells, monocytes, dendritic cells, platelets) with cluster-marker validation, and establish a baseline reference for downstream perturbation studies.\nMethod overview: PBMCs isolated from 8 mL fresh whole blood by SepMate density gradient, washed in 1× PBS + 0.04% BSA, counted on Countess II to 1,200 cells/µL viable. Target 10,000 cells loaded onto Chromium Next GEM Chip G, paired with Single Cell 3′ v3.1 Gel Beads. GEM-X partitioning on Chromium Controller, RT in droplet, cleanup, cDNA amplification, library construction per 10x CG000204 user guide. Library QC on BioAnalyzer HSDNA + Qubit dsDNA HS. Pooled and sequenced on NovaSeq 6000 SP flow cell (R1 28 bp barcode + UMI, R2 90 bp cDNA insert). Demultiplexed BCL → FASTQ via bcl2fastq, aligned + counted via Cellranger count v8.0 against GRCh38. Loaded into Seurat v5 for QC filtering (200-7500 genes/cell,",
"extra_fields": {
"Sample": {
"Tissue": {
"type": "select",
"value": "PBMC, whole blood, dissociated tumor, brain, lung, intestine, organoid"
},
"Donor": {
"type": "text",
"value": "Donor 7 (healthy)"
},
"Dissociation": {
"type": "select",
"value": "density gradient, mechanical, enzymatic (Collagenase + DNase)"
}
},
"Capture & Library": {
"Chemistry": {
"type": "select",
"value": "10x 3' v3.1, 10x 5' v2, 10x multiome, 10x fixed RNA, 10x multiplex"
},
"Target cells": {
"type": "number",
"value": "10000"
},
"Captured cells": {
"type": "number",
"value": "10182"
},
"Doublet rate": {
"type": "number",
"value": "9.0 %"
}
},
"Sequencing & QC": {
"Mean reads/cell": {
"type": "number",
"value": "28124"
},
"Median genes/cell": {
"type": "number",
"value": "1850"
},
"Mito fraction": {
"type": "number",
"value": "1.2 %"
},
"Saturation": {
"type": "number",
"value": "92 %"
},
"Clusters": {
"type": "number",
"value": "8"
}
}
},
"attached_files": [
{
"filename": "cellranger_websummary.html",
"type": "docx",
"size": "1.4 MB"
},
{
"filename": "seurat_qc_plots.pdf",
"type": "pdf",
"size": "892 KB"
},
{
"filename": "cluster_markers.csv",
"type": "csv",
"size": "48 KB"
}
],
"steps": [
{
"n": 1,
"description": "Receive 8 mL fresh whole blood from donor 7, transfer to SepMate tubes",
"duration": "0:10",
"complete": true,
"note": "IRB approval 2023-PBMC-014"
},
{
"n": 2,
"description": "PBMC isolation: SepMate density gradient, 1200×g × 10 min, wash 2× in 1× PBS + 0.04% BSA",
"duration": "0:40",
"complete": true,
"note": "Viability 96% by trypan"
},
{
"n": 3,
"description": "Count viable cells on Countess II, resuspend to 1,200 cells/µL for ~16k cells loading",
"duration": "0:08",
"complete": true,
"note": "Final: 1,220 cells/µL, 95% viability"
},
{
"n": 4,
"description": "Prepare master mix + load Chromium Next GEM Chip G with target 10,000 cells per channel",
"duration": "0:15",
"complete": true,
"note": "Chip lot 2026-04-CG · Single Cell 3' v3.1"
},
{
"n": 5,
"description": "Run Chromium Controller for GEM-X partitioning + droplet generation",
"duration": "0:12",
"complete": true,
"note": "Controller run 06:04:09:42"
},
{
"n": 6,
"description": "Reverse transcription in droplets (53°C × 45 min), cleanup, cDNA amplification (12 cycles)",
"duration": "4:00",
"complete": true,
"note": "Post-cDNA Qubit 41 ng/µL"
},
{
"n": 7,
"description": "Library construction per 10x CG000204: fragmentation, adapter ligation, sample index PCR",
"duration": "3:30",
"complete": true,
"note": "Library QC: BioAnalyzer peak 450 bp · Qubit 18 ng/µL"
},
{
"n": 8,
"description": "Pool with 3 other libraries (equimolar), submit to seq core for NovaSeq SP flow cell",
"duration": "0:30",
"complete": true,
"note": "Target 300M reads / 4 libraries = 75M/library"
},
{
"n": 9,
"description": "BCL→FASTQ via bcl2fastq, Cellranger count v8.0 alignment + UMI quantification (GRCh38)",
"duration": "12:00",
"complete": true,
"note": "10,182 cells passed · 28k reads/cell · 92% saturation"
},
{
"n": 10,
"description": "Seurat v5 QC + clustering + Azimuth annotation, archive .rds + cluster marker tables",
"duration": "4:00",
"complete": true,
"note": "8 clusters annotated · CD4/CD8 T, NK, B, Mono, mDC, pDC, Platelet"
}
],
"spreadsheet": [
[
"Cluster",
"Cell type",
"N cells",
"Marker genes",
"Notes"
],
[
"0",
"CD4 T cell",
"2444",
"CD3D, CD4, IL7R, CCR7",
"Naive + memory mixed"
],
[
"1",
"CD8 T cell",
"1833",
"CD3D, CD8A, GZMA, GZMK",
"Includes effector + memory"
],
[
"2",
"NK cell",
"1222",
"GNLY, NKG7, KLRD1, NCAM1",
"CD56-bright + CD56-dim mixed"
],
[
"3",
"B cell",
"1629",
"CD19, CD79A, MS4A1, CD37",
"Naive B dominant"
],
[
"4",
"Classical Monocyte",
"1527",
"CD14, LYZ, S100A8, S100A9",
"CD14++ CD16-"
],
[
"5",
"mDC",
"407",
"CD1C, FCER1A, HLA-DQB1",
"Conventional DC2"
],
[
"6",
"pDC",
"204",
"IL3RA, LILRA4, IRF7, GZMB",
"Plasmacytoid DC"
],
[
"7",
"Platelet",
"916",
"PPBP, GP9, ITGA2B, PF4",
"Doublet-like, kept after manual review"
]
],
"links": {
"experiments": [
{
"ref_id": "EXP-2026-0530",
"title": "Donor 7 IRB consent + sample chain of custody",
"owner": "R. Tanaka",
"date": "May 30"
},
{
"ref_id": "EXP-2026-0612",
"title": "CD8 T trajectory analysis (Monocle3)",
"owner": "R. Tanaka",
"date": "Jun 12"
}
],
"resources": [
{
"ref_id": "SOP-0438",
"title": "scRNA-Seq 10x 3' v3.1 SOP v1.8",
"owner": "QA Team",
"date": "Apr 19"
},
{
"ref_id": "CG000204",
"title": "10x Chromium Next GEM Single Cell 3' v3.1 user guide",
"owner": "10x Genomics",
"date": "Feb 7"
},
{
"ref_id": "PIPE-SC1",
"title": "Seurat v5 analysis pipeline reference docs",
"owner": "Bioinformatics",
"date": "Jan 22"
}
]
},
"compounds": [
{
"name": "Chromium Next GEM Chip G",
"catalog_lot": "1000127 · Lot 2026-04-CG",
"stock_status": "In stock"
},
{
"name": "Single Cell 3' v3.1 Gel Beads",
"catalog_lot": "1000122 · Lot 2026-04-GB",
"stock_status": "In stock"
},
{
"name": "Library Construction Kit",
"catalog_lot": "1000190 · Lot 2026-04-LC",
"stock_status": "In stock"
},
{
"name": "Dual Index Kit TT Set A (sample indices)",
"catalog_lot": "1000215 · Lot 2026-04-DI",
"stock_status": "In stock"
},
{
"name": "NovaSeq SP Flow Cell + Reagent Kit",
"catalog_lot": "20040326 · Lot 2026-05-NV",
"stock_status": "In stock"
},
{
"name": "SepMate-50 IVD tubes (PBMC isolation)",
"catalog_lot": "85460 · Lot SLBT2842",
"stock_status": "Low"
}
],
"storage": [
{
"name": "PBMC cryovial archive (donor 7, 2 × 10⁷ cells/vial)",
"location": "LN₂ vapor · Tank 1 · Rack 8 · Box PBMC-2026-D7",
"count": "×4"
},
{
"name": "cDNA + library aliquots (sequencing-grade)",
"location": "-20 °C · Freezer A-4 · Drawer 2 · Box SC-2026-06",
"count": "×3"
},
{
"name": "BCL + FASTQ + Cellranger output",
"location": "Cloud · /scrnaseq/2026-06/donor7/",
"count": "×1"
}
],
"permissions": {
"visibility": [
{
"type": "Team",
"name": "Tanaka Lab"
},
{
"type": "Person",
"name": "R. Tanaka"
}
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"can_write": [
{
"type": "Person",
"name": "R. Tanaka"
},
{
"type": "Person",
"name": "Dr. E. Kim"
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}
}