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Uropod formation on T cells, 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.

Uropod formation of T cells​

PBMCs were first stimulated into PHA blasts with PHA-L for 48 hours, washed twice in PBS and incubated with 10 ng/ml of IL2 in RPMI media for 5 days at 37°C. Approximately 300,000 PHA blasts were added to each of the 3 coated plates with 5 µg/ml of Human ICAM-1 (CD54) His-tag Fc Chimera Recombinant Protein (Catalog # A42523) for two hours. Three aliquots of cells were adhered and two of those were treated with a different cytokine each in solution, either 10 ng/ml of RANTES or MCP1, at 37°C for 1 h. The adhered cells were then fixed with 1% PFA, before being brought back into suspension via incubation with TrypLE enzyme solution (ThermoFisher) for 10 min at 37°C. For the solution conditions (control), PHA blasts were instead suspended alone or incubated in solution with either 10 ng/ml of RANTES or MCP1, followed by PFA fixation.

Cell fixation and AOC staining​

Cells adhered or in suspension were 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.

info

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)
Uropod formation on T cells v1.0 CSV2.03 KiBc3d852f5e2a72c282827eae4b2ff5973
Input filesSizeChecksum (MD5)
Uropod_CD54_fixed_R1 FASTQ1.83 GiBa5a0d6f3ff125673ea49a51f8d41d077
Uropod_CD54_fixed_R2 FASTQ2.84 GiB2272e4fcbd68c75fcb67761f5d20c8fb
Uropod_CD54_fixed_RANTES_stimulated_R1 FASTQ2.11 GiB528eb67df5ba390b21ee22b7b8f0761c
Uropod_CD54_fixed_RANTES_stimulated_R2 FASTQ3.27 GiBff720331c75ae1561d39a29d3c280ed4
Uropod_CD54_fixed_MCP1_stimulated_R1 FASTQ1.86 GiB7c3e7bbf2d27b4d1b328c2a8e64ccb0e
Uropod_CD54_fixed_MCP1_stimulated_R2 FASTQ2.88 GiBd2d3c40153f81ad59071ecf5f2803d42
Uropod_control_R1 FASTQ2.2 GiB79a143068c25e690078cc0e11ce9b5aa
Uropod_control_R2 FASTQ3.44 GiB25e1c327323685da4cc3b92ba7c06c4e
Uropod_control_RANTES_stimulated_R1 FASTQ985.49 MiB8945e215a08a10eb457a202216fc6c69
Uropod_control_RANTES_stimulated_R2 FASTQ1.49 GiB1d46465af1cad064dfb6fdf4dd7de0ce
Uropod_control_MCP1_stimulated_R1 FASTQ1.87 GiBe7ae0a04b2d8c4e329b4f54cc463ae8e
Uropod_control_MCP1_stimulated_R2 FASTQ2.98 GiB6aee46497f6dff80230876f3c55785ac
Output filesSizeChecksum (MD5)
Uropod_CD54_fixed PXL249.42 MiB2c1a7de33470d935a057466d32ef237a
Uropod_CD54_fixed_RANTES_stimulated PXL242.61 MiB8b931e7fcbb3cb7a83afb84846f1fa0b
Uropod_CD54_fixed_MCP1_stimulated PXL238.61 MiB6cae098ed9caaba6d447ab32d8c4388d
Uropod_control PXL242.06 MiBd9ef7ea8262736b87c5e91ec91326c3e
Uropod_control_RANTES_stimulated PXL116.16 MiBc2bb871a9d204b259c7026e40beb8341
Uropod_control_MCP1_stimulated PXL200.24 MiB30ec1a9e5e2ab5b63c9e91ddd0b6a40f
Report filesSizeChecksum (MD5)
Uropod_CD54_fixed HTML3.07 MiBc9f50403e2ecb5b3360c9ae714aa5b2c
Uropod_CD54_fixed_RANTES_stimulated HTML3.04 MiBe99c5d88c4aff8e5da2fb5955a1587f0
Uropod_CD54_fixed_MCP1_stimulated HTML3.06 MiB1d6e12100627ede2bb67a3bdffddc670
Uropod_control HTML3.07 MiB0a8de33ed9c3e744318c9b3001fc6730
Uropod_control_RANTES_stimulated HTML3.01 MiB6a945c737bfabf2eec06cba232aaa685
Uropod_control_MCP1_stimulated HTML3.05 MiBa230e3d28bd2137cd2e3b2f2726d50e7

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.