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Technote comparison of MPX, SCSP v1.0 vs v2.0 Kit, Immunology Panel I and II

This dataset is the basis of the technical comparison of MPX V1 vs V2. The Human Immunology panel I and panel II, samplesheets, 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 isolated from a buffy coat by Lymphoprep™️ density gradient centrifugation followed by red blood cell lysis using eBioscience™️ 1X RBC Lysis Buffer for 15 minutes. The platelet fraction was reduced by 2 repeated centrifugation steps at 100 x g for 10 minutes.

The buffy coat, drawn from a healthy volunteer with informed consent and withheld sample identity, was purchased from a Karolinska Hospital blood bank.

PHA stimulation of PBMC cells​

Half of the PBMCs were fixed as resting cells whereas half were stimulated into PHA blasts using complete RPMI media supplemented with PHA-L for 72 hours.

Cell fixation and AOC staining​

Cells in suspension were fixed with a solution of 1% v/v PFA in PBS for 15 min at room temperature. Cells were then washed once using Wash Buffer (PP016), followed by a 15 min incubation in Blocking Buffer (PP009) at 4°C. Cells were washed a final time and frozen in FBS containing 5% DMSO.

Both fixed resting and PHA-activated cells were thawed, washed once and then split into two fractions. Half of the cells were stained for 45 min at 4°C in 50 µl containing the 80-plex AOC Panel from the Human Immunology Panel I (PP010), and half of the cells were stained in the 84-plex AOC Panel from the Human Immunology Panel II (PP043). After 4 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 the 2nd Antibody Solution (PP028), followed by two washing steps in Wash Buffer, before proceeding with the MPX workflow.

MPX workflow​

Libraries were generated from cells in suspension (~20,000 cells per sample) as described in the MPX Single Cell Spatial Proteomics User Guide (v1.02) Immunology Panel I, Human (PXGIMM001) for MPX v1 or in the MPX Single Cell Spatial Proteomics User Guide (v1.00) Immunology Panel II, Human (PXGIMM002) for MPX v2.

NGS Library​

PCR was performed on 1000 cells per sample for a total of 13 cycles.

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 v0.18.0 with default parameters.

Summary Report

View summary report with quality metrics and dashboards

File Download​

info

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
Human Immunology Panel II CSV3.16 KiB23d202e7bc2fd9c3aa472e5655b5d769
SamplesheetSizeChecksum (MD5)
MPX on PBMC and PHA-stimulated cells v1.0 CSV1.19 KiBa60673c303e600ff8c1fac57266e5c22
MPX on PBMC and PHA-stimulated cells v2.0 CSV1.19 KiBeb0b0971877472ea2a81573e443eeb13
Input filesSizeChecksum (MD5)
MPX v1 on unstimulated Human PBMCs Sample01_R1 replicate 1 FASTQ2.84 GiBa77fd88eaa4a4f9e08726f428c29e401
MPX v1 on unstimulated Human PBMCs Sample01_R2 replicate 1 FASTQ4.45 GiBd38058d7c18c083ab11cf127fac9afdd
MPX v1 on unstimulated Human PBMCs Sample02_R1 replicate 2 FASTQ2.97 GiB7ef76c50a0e0aca9f889563710e83550
MPX v1 on unstimulated Human PBMCs Sample02_R2 replicate 2 FASTQ4.66 GiBb7d618efc1d1a2fef39dcde541a3b07c
MPX v1 on PHA-stimulated Human PBMCs Sample03_R1 replicate 1 FASTQ3.23 GiB7799a4c016e0a976a0b575d83c75257a
MPX v1 on PHA-stimulated Human PBMCs Sample03_R2 replicate 1 FASTQ5.06 GiBa42b5bb1fc7d712846c9778b8f5d47e7
MPX v1 on PHA-stimulated Human PBMCs Sample04_R1 replicate 2 FASTQ2.98 GiBf92943ee37db6c0f4fd003c18fafbfb5
MPX v1 on PHA-stimulated Human PBMCs Sample04_R2 replicate 2 FASTQ4.7 GiB26ea0fdc1c39d455d36fa5d74540b9be
MPX v2 on unstimulated Human PBMCs Sample05_R1 replicate 1 FASTQ3.06 GiBb191398018129c3740af83297a3988d9
MPX v2 on unstimulated Human PBMCs Sample05_R2 replicate 1 FASTQ5.01 GiB533551490f9b2b3ee0ef8518df4485b1
MPX v2 on unstimulated Human PBMCs Sample06_R1 replicate 2 FASTQ3.11 GiB95ac2298b680fe1a127a4674eecd4b59
MPX v2 on unstimulated Human PBMCs Sample06_R2 replicate 2 FASTQ5 GiBe30d713e7a848d3c2586310d8dfb81dc
MPX v2 on PHA-stimulated Human PBMCs Sample07_R1 replicate 1 FASTQ3.03 GiBb1a938b83613151834871fb7e44dbab2
MPX v2 on PHA-stimulated Human PBMCs Sample07_R2 replicate 1 FASTQ4.78 GiB344f56d5dac7f50a9ebdf04f2b9dcc24
MPX v2 on PHA-stimulated Human PBMCs Sample08_R1 replicate 2 FASTQ3.27 GiB889386320a36be649ce3e02735a8036f
MPX v2 on PHA-stimulated Human PBMCs Sample08_R2 replicate 2 FASTQ5 GiB42f5e621b28c47c7b95cae110af2e408
Output filesSizeChecksum (MD5)
MPX v1 unstimulated Human PBMCs, replicate 1, Sample01 PXL498.87 MiB95fd0113df439e111a02ac61e9948c5f
MPX v1 unstimulated Human PBMCs, replicate 2, Sample02 PXL492.03 MiB67f39ef7ef210dedaa94c9c748cd26d5
MPX v1 PHA-stimulated Human PBMCs, replicate 1, Sample03 PXL1 GiBd285e67bc0bb7e76df473262d80e3127
MPX v1 PHA-stimulated Human PBMCs, replicate 2, Sample04 PXL928.1 MiBfda1b632769fae5f3b75da593b54b0b0
MPX v2 unstimulated Human PBMCs, replicate 1, Sample05 PXL924.81 MiB229770eafd9f647b3e0f37329511c0fa
MPX v2 unstimulated Human PBMCs, replicate 2, Sample06 PXL934.65 MiB455312bb623e45fd450b5f4c7bf1cce4
MPX v2 PHA-stimulated Human PBMCs, replicate 1, Sample07 PXL1.07 GiBfefdba1ada718793c632169d09c8a8fc
MPX v2 PHA-stimulated Human PBMCs, replicate 2, Sample08 PXL1.13 GiBdf9f67d340595ee5187c8b02bcaa80dd
Report filesSizeChecksum (MD5)
MPX v1 unstimulated Human PBMCs, replicate 1, Sample01 HTML3.08 MiBa784c86e1949dd7f356913004bb1e74a
MPX v1 unstimulated Human PBMCs, replicate 2, Sample02 HTML3.08 MiB9199e401cea655e8d3966497e30b4bbb
MPX v1 PHA-stimulated Human PBMCs, replicate 1, Sample03 HTML3.15 MiB64859ffa083e77d2e7449c83f7fc310d
MPX v1 PHA-stimulated Human PBMCs, replicate 2, Sample04 HTML3.13 MiBac3e0c10bf22ae9fb965d33a3c52ee81
MPX v2 unstimulated Human PBMCs, replicate 1, Sample05 HTML3.17 MiB9270a0bd0182c171d470d57df86d634e
MPX v2 unstimulated Human PBMCs, replicate 2, Sample06 HTML3.17 MiBa1b90d060039a18efad930110128ef5a
MPX v2 PHA-stimulated Human PBMCs, replicate 1, Sample07 HTML3.16 MiB52a6321bf154f74b1c662a7d9be52c8b
MPX v2 PHA-stimulated Human PBMCs, replicate 2, Sample08 HTML3.17 MiBcd9f11e035eafd3e542b68c651b47e6b

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.