Single-cell RNA sequencing of 76,535 capillary PBMCs from 3 donors across 28 samples, freshly isolated or on ice for 24h with manual 4-layer annotation hierarchy

Sample Procurement

Samples were collected directly from participants as part of ImYoo's "Single-cell immune profiling from self-collected capillary blood" study, approved by Advarra IRB (Protocol #Pro00057361).

Sample Processing

Whole capillary blood samples were self-collected from participants using the TAP II device. Samples were either processed shortly after collection, or if being stored for longer than 4 hours, were kept in a styrofoam cooler with ice packs. Cells were isolated using EasySep Direct Human PBMC Isolation Kit (STEMCELL Technologies Catalog #19654) and cryopreserved using CryoStor CS10 (STEMCELL Technologies Catalog #07930). Upon thawing, samples were labeled in accordance with the MULTI-seq protocol (https://www.nature.com/articles/s41592-019-0433-8) and then processed on a 10X Genomics Chromium, using either the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics Product Code 1000269) or Chromium Next GEM Single Cell 3’ HT Kit v3.1 (10X Genomics Product Code 1000370). DNA libraries were sequenced on either a NovaSeq 6000 or NextSeq 550.

Data Processing

Transcriptomic sequencing data was processed using Cell Ranger v7.0.1 with default parameters. Multiplexing oligo sequencing data was processed through a custom python script that counts the number of occurrences of each sample barcode sequence and assigns it to the corresponding cell barcode. Samples were demultiplexed using a custom algorithm that estimates the background sample barcode counts, and assigns each cell a probability of belonging to each sample. Cell typing was done as part of a larger dataset and consisted of iterative manual assignments of clusters to cell types. For each cell subtype detected, a new model was trained on just the cells of that type, and the process was repeated.

Metadata Fields

• barcode: Original chromium cell barcode
• Sample IDs: Unique ID for the experimental sample that was processed with 10x Chromium, could have come from the same biological sample (identified by original_sample_id)
• Participant IDs: Unique ID for participant (here there are three participants: 2, 3 and 51)
• Cell Barcoding Runs: Unique ID for the 10x Chromium cell barcoding run in which that sample was processed. Multiple samples can be processed in a cell barcoding run.
• Lane: ID of which Chromium chip lane the cell came from
• extraction_protocol: How the PBMCs were isolated from whole blood. In this dataset all samples were processed with the TAP device.
• sample_processing_delay_seconds: The amount of time (in seconds) between when the blood was extracted from the participant and when PBMC isolation + cryopreservation was performed
• cell_barcoding_delay_days: How long PBMC samples were stored in liquid nitrogen prior to being thawed and processed on 10x
• cell_barcoding_protocol: Which single cell RNA sequencing experimental protocol was used. Here all samples were processed with 10x v3.1 chemistry.
• run_lane_batch: Concatenation of columns Cell Barcoding Runs and Lane to provide a unique ID for experimental processing batch (i.e. the DNA library)
• cell_type_level_1: Level 1 of a 4-tier PBMC ontology that does not provide a label for low quality cells - those were left as NaNs.
• cell_type_level_2: Level 2 of a 4-tier PBMC ontology that does not provide a label for low quality cells - those were left as NaNs.
• cell_type_level_3: Level 3 of a 4-tier PBMC ontology that does not provide a label for low quality cells - those were left as NaNs.
• cell_type_level_4: Level 4 of a 4-tier PBMC ontology that does not provide a label for low quality cells - those were left as NaNs.
• c1: Level 1 of a 4-tier PBMC ontology that also provides label for low quality cells, such as Debris, Doublets, experimental artifacts and others. These additional labels can be used for creating a junk detector.
• c2: Level 1 of a 4-tier PBMC ontology that also provides label for low quality cells, such as Debris, Doublets, experimental artifacts and others. These additional labels can be used for creating a junk detector.
• c3: Level 1 of a 4-tier PBMC ontology that also provides label for low quality cells, such as Debris, Doublets, experimental artifacts and others. These additional labels can be used for creating a junk detector.
• c4: Level 1 of a 4-tier PBMC ontology that also provides label for low quality cells, such as Debris, Doublets, experimental artifacts and others. These additional labels can be used for creating a junk detector.
• original_sample_id: Some samples were derived from the same originating whole blood sample. This field specifies the source of the whole blood sample.