Longitudinal single-cell RNA sequencing of 55,260 capillary PBMCs from 2 donors with ME/CFS before, during, and after antibiotic treatment

<p><strong>Sample Procurement</strong></p> <p>Samples were self-collected directly from participants as part of <a href="https://www.remissionbiome.org/" target="_blank" rel="noopener">RemissionBiome</a>'s pilot study, and processed by <a href="https://www.imyoo.health/">ImYoo</a>. For additional details on the experiment, see&nbsp;<a href="https://www.imyoo.health/post/n-of-1-studies-are-scaling-and-they-re-making-discoveries-in-the-toughest-diseases" target="_blank" rel="noopener">ImYoo's&nbsp;blog post</a> and corresponding comments.</p> <p><strong>Sample Processing</strong></p> <p>Whole capillary blood samples were self-collected from participants using the&nbsp;<a href="https://yourbiohealth.com/en-us/virtually-painless-blood-collection-devices-for-clinical-trials-and-wellness-testing">TAP II</a> device. Samples were kept in a temperature stable thermos or styrofoam cooler with ice packs and shipped with overnight shipping to ImYoo labs. Cells were isolated using&nbsp;<a href="https://www.stemcell.com/products/easysep-direct-human-pbmc-isolation-kit.html">EasySep Direct Human PBMC Isolation Kit</a>&nbsp;(STEMCELL Technologies Catalog #19654) and cryopreserved using&nbsp;<a href="https://www.stemcell.com/cryostor-cs10.html">CryoStor CS10</a>&nbsp;(STEMCELL Technologies Catalog #07930). Upon thawing, samples were labeled in accordance with the MULTI-seq&nbsp;protocol (<a href="https://www.nature.com/articles/s41592-019-0433-8">https://www.nature.com/articles/s41592-019-0433-8</a>) and then processed on a 10X Genomics Chromium, using the Chromium Next GEM Single Cell 3&rsquo; HT Kit v3.1 (10X Genomics Product Code 1000370). DNA libraries were sequenced on a Complete Genomics DNBSEQ-G400 sequencer.</p> <p><strong>Data Processing</strong></p> <p>Transcriptomic sequencing data was processed using Cell Ranger v7.0.1 with default parameters. Multiplexing oligo sequencing data was processed through a custom python script that counts the number of occurrences of each sample barcode sequence and assigns it to the corresponding cell barcode. Samples were demultiplexed using a custom algorithm that estimates the background sample barcode counts, and assigns each cell a probability of belonging to each sample. Cell typing was done by training an <a href="https://scvi-tools.org/" target="_blank" rel="noopener">scVI model</a> on all cells with default parameters, and "library" as the batch_key. The latent space was then used to perform leiden clustering, and clusters were mapped to cell types based on common marker gene expression.</p> <p><strong>Differential Expression</strong></p> <p>Differential expression was calculated using DEseq2. For each cluster and cell type, cells from the same sample in the same participant were summed together to create a pseudocell, and passed as pseudobulked samples into DESeq2 to compare "Event" samples to "Baseline" samples.</p> <p><strong>Metadata Fields</strong></p> <ul> <li><strong>barcode:</strong> 10X cell barcode</li> <li><strong>sample_id:</strong>&nbsp;Unique ID for the experimental sample that was processed with 10x Chromium, could have come from the same biological sample (identified by&nbsp;<strong>source_sample_id</strong>)</li> <li><strong>condition</strong>: Whether this sample is before ("Baseline"), during ("Event"), or after ("New Baseline") the antibiotic intervention</li> <li><strong>participant_id:</strong> Unique ID for participant. There are two participants: P363, and P364. P363 experienced a full remission event, while P364 did not.</li> <li><strong>extracted_on</strong>: The date and time the blood was extracted</li> <li><strong>cell_barcoding_run_id:</strong>&nbsp;Unique ID for the 10x Chromium cell barcoding run in which that sample was processed. Multiple samples can be processed in a cell barcoding run.</li> <li><strong>extraction_type</strong>: Whether the PBMCs were isolated from Venous or Capillary blood</li> <li><strong>lane:</strong>&nbsp;ID of which Chromium chip lane the cell came from</li> <li><strong>sample_processing_delay_seconds:</strong>&nbsp;The amount of time (in seconds) between when the blood was extracted from the participant and when PBMC isolation + cryopreservation was performed</li> <li><strong>cell_barcoding_delay_days:</strong>&nbsp;How long PBMC samples were stored in liquid nitrogen&nbsp;prior to being thawed and processed on 10x</li> <li><strong>cell_barcoding_protocol</strong>: Which single cell RNA sequencing experimental protocol was used. Here all samples were processed with 10x v3.1 chemistry.</li> <li><strong>library:</strong>&nbsp;Concatenation of columns&nbsp;<strong>Cell Barcoding Runs</strong>&nbsp;and&nbsp;<strong>Lane</strong>&nbsp;to provide a unique ID for experimental processing batch (i.e. the DNA library)</li> <li><strong>leiden:</strong> The cluster this cell belongs to after automatic cell clustering</li> <li><strong>cell_type:</strong> Manually labeled cell type</li> <li><strong>source_sample_id:</strong> Some samples may be derived from the same originating whole blood sample. This field specifies the source of the whole blood sample.</li> </ul>