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1k 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.

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)
1k Human PBMCs v1.0 CSV754 B2afd82ce673a3e22bbfe5986c956ea3d
Input filesSizeChecksum (MD5)
Unstimulated Human PBMCs Sample01_R1 FASTQ858.34 MiBc16618a5b0e224193d48ccd3098ad665
Unstimulated Human PBMCs Sample01_R2 FASTQ1.28 GiBf595acb2e3f848bc2ef0fd3afda1c3b8
Unstimulated Human PBMCs Sample02_R1 FASTQ956.87 MiBbae116ba54b523defdc2b6f144eda449
Unstimulated Human PBMCs Sample02_R2 FASTQ1.42 GiBb4d1b2c311950108f1d7ed6ae7f5616c
Output filesSizeChecksum (MD5)
Unstimulated Human PBMCs Sample01 PXL185 MiB9a597b1555336477bbefba86b88c8121
Unstimulated Human PBMCs Sample02 PXL256.27 MiB977deee49f45e07d4dfeafcd0fb848a7
Report filesSizeChecksum (MD5)
Unstimulated Human PBMCs Sample01 HTML3.02 MiB7aa7216e36e1e6e39a068d58b950c475
Unstimulated Human PBMCs Sample02 HTML3.06 MiB5ab5ea568e9843e1aa429cb162186d9d

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.