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CD3 Stimulated Human PBMCs, SCSP v1.0 Kit, Immunology Panel I

This dataset is part of the Molecular Pixelation (MPX) publication. The Human Immunology panel I, samplesheet, and MPX library FASTQ files are provided below, as well as the Pixelator output files.

PBMC extraction from whole-blood​

Human peripheral blood mononuclear cells (PBMCs) were separated from whole-blood collected in heparin or EDTA blood collection tubes by Ficoll-Paque density gradient centrifugation. The platelet fraction was reduced by 3 repeated centrifugation steps at 100 x g for 10 minutes.

PBMC samples were purchased from a Karolinska Hospital blood bank drawn from healthy volunteers with informed consent and withheld sample identity or other medical information.

CD3 capping of PBMCs​

PBMCs were first incubated with 50 µg/ml of human IgG for 15 min at 4°C to block Fc receptors and then incubated with 20 µg/ml of anti-CD3 AOC for 40 min at 4°C. After two washes, cells were incubated with 40 µg/ml of Goat anti-mouse secondary antibody for 40 min at 4°C, followed by incubation at 37°C for 1 hour. The capped cells were then immediately fixed with PFA.

Cell fixation and AOC staining​

Cells were re-suspended in PBS and fixed with a solution of 1% v/v PFA in PBS for 15 min at room temperature. Cells were washed once in PBS, followed by addition of a Blocking/Quenching buffer in the PBS solution. Finally, cells were incubated for 15 min at 4°C, followed by a wash in PBS to remove the blocking/quenching solution.

Fixated and blocked cells were stained for 30 min at 4°C in a 50 µl reaction containing a cocktail of 80 AOCs from the Human Immunology Panel I, each at a concentration of 5 µg/ml, in a staining buffer. After 3 washing steps in Wash Buffer, AOCs bound to cells were stabilized using a secondary antibody by incubating the cells for 30 min at 37°C in a secondary antibody solution consisting of 20 µg/ml secondary antibody, followed by two washing steps in Wash Buffer, before proceeding with the MPX workflow.

MPX workflow​

Libraries were generated from cells in suspension (~15,000 cells per sample) as described in the MPX Single Cell Spatial Proteomics User Guide (v1.01) Immunology Panel I, Human (PXGIMM001).

NGS Library​

PCR was performed in a 40 µl PCR reaction containing 1x Q5 HotStart Hifi PCR master mix (New England Biolabs), 0.4 µM of Illumina adapter PCR primers (ILM_p5_PCR, ILM_p7_PCR) containing 8 nt sample indexes to allow multiplexing and 15 µl of sample from the lambda exonuclease step.

The PCR products were purified twice using AmpureXP SPRI beads (Beckman-Coulter) according to manufacturer’s instructions and subsequently quantified using Qubit HsDNA assay (ThermoFisher).

Sequencing​

The purified PCR products were diluted to 0.65 nM with 15% PhiX spiked in and paired-end sequenced on an Illumina sequencing system, using 44 cycles for read1 and 78 cycles for read2.

  • Instrument: Illumina NextSeq2000
  • Read 1: 44 cycles (25 bp UPIB, 19 bp PBS2)
  • Read 2: 78 cycles (8 bp Antibody BC, 10 bp UMI, 22 bp PBS1, 25 bp UPIA, 13 bp PBS2)
  • i5 index: 8 cycles (sample index)
  • i7 index: 8 cycles (sample index)

Data Processing​

MPX sequencing data was processed by Pixelator v0.19.0 with default parameters.

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This dataset from the MPX method paper has been updated and reprocessed with the latest Pixelator version.

File Download​

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In order to process the dataset FASTQ files, you need to have the required infrastructure by nf-core/pixelator.

Panel fileSizeChecksum (MD5)
Human Immunology Panel I CSV3.79 KiB692b356a9b1fff3d7b00590a12d5dd13
SamplesheetSizeChecksum (MD5)
CD3 Stimulated Human PBMCs v1.0 CSV1.43 KiBcd05a7230a2a3bd6fb4790051e26c88d
Input filesSizeChecksum (MD5)
Sample07_R1 FASTQ977.37 MiB4a44a850838612765b4976c933a0fae7
Sample07_R2 FASTQ1.44 GiBe0585a454276e9e6d63bebaaba3396e6
Sample08_R1 FASTQ1.07 GiB843f793eb7ee5a8c99528fe5d3125922
Sample08_R2 FASTQ1.6 GiB203a88eaf9e05979e057fb11f46e5b95
Output filesSizeChecksum (MD5)
CD3 stimulated Sample07 PXL217.03 MiB59bb053708ec14afa6e306a03a4c83a0
CD3 stimulated Sample08 PXL238.41 MiB455306629a499dbead206f9ec19319d2
Report filesSizeChecksum (MD5)
CD3 stimulated Sample07 HTML3.05 MiB8d0e496724d3410ee95da75254e7215b
CD3 stimulated Sample08 HTML3.06 MiBee30cfa55beffe6dd7de007f39b9a0af

License​

Creative Commons Attribution-ShareAlike 4.0 International (CC BY-SA 4.0)

How to Cite​

If you are using this data in your research, please cite the original MPX publication as follows:

**Molecular pixelation: spatial proteomics of single cells by sequencing.**Filip Karlsson, Tomasz Kallas, Divya Thiagarajan, Max Karlsson, Maud Schweitzer, et al.Nature Methods, 2024 May 8, doi: https://doi.org/10.1038/s41592-024-02268-9.