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Rituximab stimulated Raji cells, SCSP v1.0 Kit, Immunology Panel I

This dataset is part of the Molecular Pixelation (MPX) publication. A modified Human Immunology panel I, containing the Rituximab antibody oligo conjugate (AOC), the samplesheet of the experiment, and the MPX library FASTQ files are provided below, as well as the Pixelator output files.

Rituximab stimulation of Raji cells​

Raji cells were Fc-receptor blocked with 50 µg/ml of human IgG for 15 min at 4 °C and washed. Cells were then either PFA fixed directly (control) or incubated with 20 µg/ml of Rituximab AOC (treated) in RPMI media for 60 min at 37 ℃, followed by washing and PFA fixation.

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 CSV with Rituximab AOC3.89 KiB84fb9b0b0cc2e4e961d0ef5c173dd945
SamplesheetSizeChecksum (MD5)
Rituximab stimulated Raji cells v1.0 CSV945 Bee710d477284fb44462f203a9dad542e
Input filesSizeChecksum (MD5)
Control Sample03_R1 FASTQ2.06 GiBd057001da3e622c44d166030c59a6dd8
Control Sample03_R2 FASTQ3.16 GiB920539e6c712fd5765db9d9d1c9f72cb
Treated Sample04_R1 FASTQ3.8 GiB12ede73ff41be6adc86546b33a45aafb
Treated Sample04_R2 FASTQ5.8 GiB6e1db1773c0d8995b65171d8cb3d8185
Output filesSizeChecksum (MD5)
Control Sample03 PXL579.41 MiBb9f4145b84aa5483c717f56ded2802a3
Treated Sample04 PXL896.61 MiBd65db61682b8496a3ffe9c9bc58a7688
Report filesSizeChecksum (MD5)
Control Sample03 HTML3.07 MiB0bbfdf7d7654d4fab544c7ddc5967a89
Treated Sample04 HTML3.12 MiB615c8c8cd01090320cae1b51af33e257

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.