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Sample GSM8139397

Query DataSets for GSM8139397

Status

Public on Nov 06, 2024

Title

FLD041FLD029FLD030 BCR library 2

Sample type

SRA

Source name

blood

Organism

Homo sapiens

Characteristics

tissue: blood
cell type: mononuclear cells (MNCs)
library type: mRNA

Extracted molecule

polyA RNA

Extraction protocol

Organ donor samples in saline or University of Wisconsin solution were placed on ice, transported to the laboratory, and processed within 2 to 4 hours of procurement. Single cell suspensions were obtained from blood, bone marrow, spleen, lungs, and associated lymph nodes (LN) using mechanical and enzymatic digestion as previously described (Nat Med 22, 72-77. 10.1038/nm.4008). Blood and bone marrow samples were diluted with wash buffer (PBS (Corning, cat #20-030-CV) + 2mM EDTA (Corning, cat #46-034-CI) + 5% FBS (GeminiBio, cat #100-106)), and mononuclear cells were isolated by density centrifugation using Ficoll-Paque PLUS (GE Healthcare, catalog no. 17-1440-03). Lymph nodes were isolated by dissection from the mesentery of the small and large intestine, the tracheobronchial tree of the lung, and inguinal fat. Tissue samples were first mechanically disrupted with scissors. Isolated LNs and lung were then placed into digestion media (IMDM + Collagenase D 1mg/mL (Millipore Sigma cat# 11088882001) and + DNAse 0.1mg/mL (Millipore Sigma cat# DN25-5G)) on a shaker at 37C for 30 minutes followed by addition of 0.5M EDTA (pH 8.0). Digested LNs and lung as well as mechanically disrupted spleen were then pushed through 100m filters and washed with buffers. The samples then underwent density centrifugation as above to isolate live leukocytes. For healthy blood samples, we obtained peripheral blood from a consenting, healthy volunteers by venous puncture. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS, following manufacturer's protocols for density gradient centrifugation. Single cell mononuclear suspensions isolated from blood and tissue samples prepared as above were plated in 96 well U-bottom plates (1X10^6 cells per well) in 200uL of complete media (RPMI (Corning, catalog no. 10-040-CM), 10% inactivated human AB serum (Gemini, catalog no. 507533010) and penicillin-streptomycin-glutamate (Thermo Fisher Scientific, catalog no. 10378016)) and incubated overnight at 37C in 5% CO2. The next day, the wells were split (5x10^5 cells per well) and topped up with complete media to 200uL. Cells were then blocked for 15 minutes with anti-CD40 monoclonal antibody (0.5g/mL) (Miltenyi Biotec, catalog no. 130-094-133). Subsequently, a SARS-CoV-2 Spike Megapool (S MP) (1g/mL), designed and synthesized as previously described (Cell 181, 1489-1501 e1415. 10.1016/j.cell.2020.05.015), was added to the cell culture for 24 hours. The S MP consists of 253 15-mer peptides overlapping by 10 residues and covering the entire S protein. As controls, additional cells were stimulated with an equimolar amount of DMSO, or a combination of 3 Influenza peptide Mega pools at 1g/mL (CD4 HA, CD4 Other, and CD8), synthesized as previously described (Curr Protoc. 2023 Nov;3(11):e934. doi: 10.1002/cpz1.934.). Following 24-hour stimulation, each tissue and stimulation condition combination for a particular donor was combined, treated with 10uL each of Trustain FcX (BioLegend) and FcR Blocking Reagent (Miltenyi) at 4C for 10 minutes, and then incubated with 2 uL (1 ug) of a unique TotalSeq-C anti-human DNA-barcoded hashtag for 30 minutes at 4C and washed 3 times with FACS buffer) as described. Subsequently all peptide-stimulated samples from a single donor were combined and all DMSO-stimulated samples from a single donor were combined. These samples were stained with BV421 labeled full length Spike protein and a panel of fluorescently labeled antibodies and washed as described above. These samples were sorted using the BD FACSAria III Cell Sorter. Peptide-stimulated samples were sorted for AIM+ CD4 T cells (Live, CD45+, CD19-, CD3+, CD4+, OX40 & 4-1BB++), AIM+ CD8 T cells (Live, CD45+, CD19-, CD3+, CD8+, CD25 & 4-1BB++), and Spike B cells (Live, CD45+, CD19+, CD3-, Spike-BV421+). DMSO-stimulated cells were sorted for AIM-CD4 T cells (Live, CD45+, CD19-, CD3+, CD4+, OX40 & 4-1BB--), AIM-CD8 T cells (Live, CD45+, CD19-, CD3+, CD8+, CD25 & 4-1BB--), and Total B cells (Live, CD45+, CD19+, CD3-, Spike-BV421-). Each sample of sorted cells was counted, and for each donor, a fraction of AIM-CD4 T, AIM-CD8 T, and Total B cells were spiked into the AIM+CD4+T, AIM+CD8+T, and Spike B cell samples to reach ~80K cells for per donor to have adequate cell numbers to manipulate for CITE-seq staining and have sufficient cell numbers after washing for input into 10x microfluidics chip (e.g. spike in cells to achieve 80k cells, yielding ~50k out post-wash for 33.3k cell requirement to achieve 20k cell capture on chip). The samples were then stained with TotalSeq C Human Universal Cocktail V1.0 and washed 3 times with FACS buffer by centrifugation or with a Curiox HT2000 Laminar Wash System (Curiox, 5 uL/sec speed, 27 cycles, 50 uL loading volume). The resultant stained cells (33.3K) were resuspended in 10x genomics recommended loading buffer (1X DPBS w/ 0.04% BSA) for loading into 10x Genomics single sequencing workflows with a target of 20K cell captured per lane on chip. To investigate the role of Tregs in the AIM assay, we performed CD25 depletion on our single cell LLN suspension from D616 prior to the above peptide stimulation and sorting protocols. For depletion, single cell suspensions were pre-incubated with 10uL each of Trustain FcX (BioLegend, catalog no. 422322) and FcR Blocking Reagent (Miltenyi, catalog no. 130-059-901) at 4C for 10 minutes followed by addition of 10uL of biotinylated anti-CD25 for 30 minutes on ice and washed with FACS buffer three times. Washed BioMag Plus Streptavidin Beads (BangsLabs catalog no. BP628) were resuspended in 100uL of FACS buffer and incubated with the single cell suspension at room temperature for 5 minutes. 1.5mL of DPBS was added to the tube and placed on the EasySep magnet (STEMCELL Technologies catalog no. 18000) for 5 minutes. The supernatant was collected and set aside. The remaining beads were combined with DPBS and placed back on the magnet for an additional 5 minutes prior to collecting the supernatant and combining with the supernatant from the first separation. This sample was then plated at 1X10^6 cells per well in 96-well U bottom plates for in vitro peptide stimulations as above. DMSO control, SARS or Flu peptide stimulated, and CD25 depleted SARS or Flu stimulated were FACS sorted with AIM+ gates described above. SARS or Flu peptide stimulated CD4 T cells were also sorted using an altenative AIM+ gating strategy (Live, CD45+, CD19-, CD3+, CD4+, OX40 & CD40LG) while maintaining original CD8 gating strategy. Stimulated and sorted single cell-suspensions were loaded onto a 5v2 high throughput (HT) Chromium Next GEM Single Cell Chip N (10x Genomics) for co-encapsulation with barcoded GelBeads with a target capture of 20-30k cells per sample.
Organ donor samples: Captured mRNA was reverse transcribed to cDNA, amplified, and converted to libraries using the Chromium Next GEM Single Cell 5 HT Reagent kit v2 (10X genomics) protocol and recommended reagents with a 5 Feature Barcode kit (10x genomics) for antibody-derived tag amplification, a Chromium Single Cell Human TCR Amplification kit (10x Genomics) for targeted TCR amplification, and a Chromium Single Cell Human BCR Amplification kit (10x Genomics) for targeted BCR amplification. The scRNA-seq gene expression (GEX) libraries, antibody derived tag (ADT) CITE-seq/hashtag libraries, scTCR-seq and, scBCR-seq libraries were sequenced on S4 flow cells (ADT, GEX libraries pooled together and TCR/BCR libraries pooled on independent flow cell lanes) with an Illumina NovaSeq 6000 (~2.5 billion, 2 x 100-bp, reads per lane). The scTCR-seq and sc-BCRseq libraries for D561 and D574 were sequenced with a 150-cycle high-output kit on an Illumina NextSeq 550 (read 1: 26 bp; read 2: 114 bp; index read 1: 10 bp; index read 2: 10 bp). Hashtag IDs for D534,D559,D561,D564,D568,D574: (HTO1: Donor blood AIM+ SARS, HTO2: Donor blood AIM+ Flu, HTO3: Donor blood AIM- DMSO control, HTO4: Donor bone marrow AIM+ SARS, HTO5: Donor bone marrow AIM+ Flu, HTO6: Donor bone marrow AIM- DMSO control, HTO7: Donor spleen AIM+ SARS, HTO8: Donor spleen AIM+ Flu, HTO9: Donor spleen AIM- DMSO control, HTO10: Donor lung AIM+ SARS, HTO12: Donor lung AIM+ Flu, HTO13: Donor lung AIM- DMSO control, HTO14: Donor lung lymph node AIM+ SARS, HTO15: Donor lung lymph node AIM+ Flu, HTO16: Donor lung lymph node AIM- DMSO control). For D561D564 library, only LNG sample is from D564, all other tissues are from D561. (BLD = blood, BM = bone marrow, SPL = spleen, LNG = lung, LLN = lung lymph node, CITE-seq = cellular indexing of transcriptomes and epitopes).
Healthy blood samples: Captured mRNA was reverse transcribed to cDNA, amplified, and converted to libraries using the Chromium Next GEM Single Cell 5 HT Reagent kit v2 (10X genomics) protocol and recommended reagents with a 5 Feature Barcode kit (10x genomics) for antibody-derived tag amplification, a Chromium Single Cell Human TCR Amplification kit (10x Genomics) for targeted TCR amplification, and a Chromium Single Cell Human BCR Amplification kit (10x Genomics) for targeted BCR amplification. The scRNA-seq gene expression (GEX) libraries, antibody derived tag (ADT) CITE-seq/hashtag libraries, scTCR-seq and, scBCR-seq libraries were sequenced on S4 flow cells (ADT, GEX libraries pooled together and TCR/BCR libraries pooled on independent flow cell lanes) with an Illumina NovaSeq 6000 (~2.5 billion, 2 x 100-bp, reads per lane). Hashtag IDs for healthy blood donors FLD032,FLD040,FLD015,FLD041,FLD029,FLD030: (HTO1: Donor 1 blood AIM+ SARS, HTO2: Donor 1 blood AIM+ Flu, HTO3: Donor 1 blood AIM- DMSO control, HTO4: Donor 2 blood AIM+ SARS, HTO5: Donor 2 blood AIM+ Flu, HTO6: Donor 2 blood AIM- DMSO control, HTO7: Donor 3 blood AIM+ SARS, HTO2: Donor 3 blood AIM+ Flu, HTO3: Donor 3 blood AIM- DMSO control). Donors 1-3 in hashtag IDs correspond to FLD-prefixed donors in corresponding order in library ID. (BLD = blood, BM = bone marrow, SPL = spleen, LNG = lung, LLN = lung lymph node, CITE-seq = cellular indexing of transcriptomes and epitopes).
Donor 616: Captured mRNA was reverse transcribed to cDNA, amplified, and converted to libraries using the Chromium Next GEM Single Cell 5 HT Reagent kit v2 (10X genomics) protocol and recommended reagents with a 5 Feature Barcode kit (10x genomics) for antibody-derived tag amplification. The scRNA-seq gene expression (GEX) libraries and antibody derived tag (ADT) CITE-seq/hashtag libraries libraries were sequenced on S4 flow cells (ADT, GEX libraries pooled together on independent flow cell lanes) with an Illumina NovaSeq 6000 (~2.5 billion, 2 x 100-bp, reads per lane). Hashtag IDs for D616: (HTOs 1,3,4,5,6,7: Donor 616 LLN AIM- DMSO control, HTO8: Donor 616 LLN AIM+ Flu peptide stimulation, HTO9: Donor 616 LLN AIM+ SARS peptide stimulation, HTO13: Donor 616 LLN CD25 depletion AIM+ Flu peptide stimulation, HTO14: Donor 616 LLN CD25 depletion AIM+ SARS peptide stimulation, HTO10: Donor 616 LLN CD40LG+/OX40+ alternate CD4 AIM+ gate Flu peptide stimulation, HTO12: Donor 616 LLN CD40LG+/OX40+ alternate CD4 AIM+ gate SARS peptide stimulation). (BLD = blood, BM = bone marrow, SPL = spleen, LNG = lung, LLN = lung lymph node, CITE-seq = cellular indexing of transcriptomes and epitopes).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

5' mRNA library: read1 file contains cell barcode and UMI; read2 file contains transcript.
filtered_contig_annotations.BCR.txt.gz

Data processing

scRNA-seq reads were analyzed by pseudoalignment using kallisto v0.46.2 (GRCh38 with Gencode v24 annotation) and bustools v0.40.0. CITE-seq and hashtag barcodes were demultiplexed and extracted using DropSeqPipeline8 (https://github.com/simslab/DropSeqPipeline8). GEX count matrix was filtered for cell barcodes with >1000 molecules/cell. These cell barcodes were used to filter ADT hashtag/CITE-seq count matrix from DropSeq8. Hashtags were demultiplexed on filtered HTO/ADT count matrix using the HashSolo (https://github.com/calico/solo/blob/master/solo/hashsolo.py) implementation in ScanPy (https://github.com/scverse/scanpy, scanpy.external.pp.hashsolo function) where prior cell frequency arguments for HashSolo function were based on theoretical doublet rate in 5’v2 HT 10x chip based on cell input (e.g. 0.08 doublet rate, 0.01 negative, 0.91 singlet rate, for 20k cell input).
Immune receptor libraries (TCR and BCR) were aligned using Cell Ranger 7.0.1 (vdj function) using reference vdj_GRCh38_alts_ensembl-7.0.0.
A mitochondrial gene filter was applied for any cells above a 95% confidence interval for MT pseudo-alignment rate on a per library basis. A hemoglobin gene filter was applied to all libraries for cells > 0.01 pseudo-alignment rate for HB genes.
MultiModal Classifier Hierarchy (MMOCHI, https://github.com/donnafarberlab/MMoCHi), which is trained using events selected by thresholding of user-defined marker genes and proteins, was implemented to define immune cell subsets across sites. MMOCHI classification legend: (CD4N = Naive CD4 T cell, CD4Treg = CD4 Regulatory T cell, CD4CM = Central Memory CD4 T Cell, CD4EM = Effector Memory CD4 T cell, CD8EM = Effector Memory CD8 T cell, CD8N = Naive CD8 T Cell, CD8CM = Central Memory CD8 T Cells, B_cell_N = Naive B Cells, B_cell_Innate_M = Innate Memory B Cells (IgD+CD27+), B_cell_CD27-IgD- = CD27-IgD- B cells, Plasma_cluster = Plasma Cells identified by GEX clustering, Myeloid_cluster = Myeloid Cells identified by GEX clustering, NK_Cluster = NK Cells identified by GEX clustering). Memory B cell classifications are subtyped by receptor isotype.
Assembly: GRCh38/Ensembl93
Supplementary files format and content: GEX and ADT libraries: tab-delimited text files with cell-identifying barcodes, hashtags, donor, tissue site, MMOCHI immune cell classification, stimulation condition (w/ applicable sort information), and count matrix
Supplementary files format and content: TCR and BCR libraries: tab-delimited text files with cell-identifying barcodes, immune receptor chain, VDJ genes, cdr3 amino acid sequence, cdr3 nucleotide sequence, raw clonotype id and other alignment statistics/metadata outputted from CellRanger.
Library strategy: CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing)

Submission date

Mar 11, 2024

Last update date

Nov 06, 2024

Contact name

Alex George

Organization name

Columbia University

Lab

Sims Lab

Street address

960 Broadway, 2nd Floor, Room 203AC