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

Sample Procurement

Samples were self-collected directly from participants as part of RemissionBiome's pilot study, and processed by ImYoo. For additional details on the experiment, see ImYoo's blog post and corresponding comments.

Sample Processing

Whole capillary blood samples were self-collected from participants using the TAP II 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 EasySep Direct Human PBMC Isolation Kit (STEMCELL Technologies Catalog #19654) and cryopreserved using CryoStor CS10 (STEMCELL Technologies Catalog #07930). Upon thawing, samples were labeled in accordance with the MULTI-seq protocol (https://www.nature.com/articles/s41592-019-0433-8) and then processed on a 10X Genomics Chromium, using the Chromium Next GEM Single Cell 3’ HT Kit v3.1 (10X Genomics Product Code 1000370). DNA libraries were sequenced on a Complete Genomics DNBSEQ-G400 sequencer.

Data Processing

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 scVI model 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.

Differential Expression

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.

Metadata Fields

barcode: 10X cell barcode

sample_id: Unique ID for the experimental sample that was processed with 10x Chromium, could have come from the same biological sample (identified by source_sample_id)

condition: Whether this sample is before ("Baseline"), during ("Event"), or after ("New Baseline") the antibiotic intervention

participant_id: Unique ID for participant. There are two participants: P363, and P364. P363 experienced a full remission event, while P364 did not.

extracted_on: The date and time the blood was extracted

cell_barcoding_run_id: 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.

extraction_type: Whether the PBMCs were isolated from Venous or Capillary blood

lane: ID of which Chromium chip lane the cell came from

sample_processing_delay_seconds: The amount of time (in seconds) between when the blood was extracted from the participant and when PBMC isolation + cryopreservation was performed

cell_barcoding_delay_days: How long PBMC samples were stored in liquid nitrogen prior to being thawed and processed on 10x

cell_barcoding_protocol: Which single cell RNA sequencing experimental protocol was used. Here all samples were processed with 10x v3.1 chemistry.

library: Concatenation of columns Cell Barcoding Runs and Lane to provide a unique ID for experimental processing batch (i.e. the DNA library)

leiden: The cluster this cell belongs to after automatic cell clustering

cell_type: Manually labeled cell type

source_sample_id: Some samples may be derived from the same originating whole blood sample. This field specifies the source of the whole blood sample.

样本采集

样本为RemissionBiome试点研究中直接从受试者处自行采集的样品，由ImYoo进行处理。如需了解实验更多细节，请参阅ImYoo的博客文章及相关评论。

样本处理

受试者使用TAP II设备自行采集全毛细血管血样。样本置于配备冰袋的恒温保温瓶或泡沫保温箱中，并通过隔夜快递运至ImYoo实验室。采用EasySep Direct人外周血单个核细胞分离试剂盒（STEMCELL Technologies 货号#19654）分离细胞，并使用CryoStor CS10（STEMCELL Technologies 货号#07930）进行冷冻保存。解冻后，样本按照MULTI-seq实验方案（https://www.nature.com/articles/s41592-019-0433-8）进行标记，随后使用10X Genomics Chromium Next GEM单细胞3’ HT试剂盒v3.1（10X Genomics 产品编号1000370）在10X Genomics Chromium系统上完成处理。DNA文库在Complete Genomics DNBSEQ-G400测序仪上进行测序。

数据处理

转录组测序数据使用Cell Ranger v7.0.1以默认参数进行处理。多重寡核苷酸测序数据通过自定义Python脚本处理，该脚本可统计每个样本标签序列的出现次数，并将其分配至对应的细胞标签。使用自定义算法对样本进行解复用：先估算背景样本标签计数，再为每个细胞分配其属于各样本的概率。细胞分型通过在所有细胞上训练scVI模型完成，采用默认参数，并以"library"作为批次键（batch_key）。随后利用潜空间进行Leiden聚类，并基于常见标记基因表达将聚类簇映射至细胞类型。

差异表达分析

使用DESeq2进行差异表达分析。针对每个聚类簇与细胞类型，将同一受试者同一样本中的细胞进行汇总，构建伪细胞，并将其作为伪批量样本输入DESeq2，以比较"Event"样本与"Baseline"样本。

元数据字段

barcode: 10X细胞标签（cell barcode）
sample_id: 经10X Chromium处理的实验样本唯一标识符，可源自同一原始生物样本（由source_sample_id标识）
condition: 样本所处的抗生素干预阶段，分为"Baseline（干预前）"、"Event（干预中）"与"New Baseline（干预后）"
participant_id: 受试者唯一标识符。本数据集共包含2名受试者：P363与P364。其中P363实现了完全缓解，P364未实现。
extracted_on: 血液采集的日期与时间
cell_barcoding_run_id: 该样本所参与的10X Chromium细胞标签实验运行唯一标识符。单次实验运行可同时处理多个样本。
extraction_type: 外周血单个核细胞（PBMC）的采集来源，分为静脉血与毛细血管血
lane: 细胞所属的Chromium芯片泳道标识符
sample_processing_delay_seconds: 从受试者体内采集血液至完成PBMC分离与冷冻保存的时长（单位：秒）
cell_barcoding_delay_days: PBMC样本在液氮中储存，直至解冻并在10X系统上完成处理的时长（单位：天）
cell_barcoding_protocol: 所用的单细胞RNA测序实验方案。本数据集所有样本均采用10X v3.1化学试剂体系
library: 将"Cell Barcoding Runs"与"Lane"两列拼接得到的实验处理批次唯一标识符（即DNA文库）
leiden: 自动细胞聚类后该细胞所属的聚类簇编号
cell_type: 人工标注的细胞类型
source_sample_id: 部分样本可源自同一原始全血样本，该字段用于标识全血样本的来源