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Sample GSM5718920

Query DataSets for GSM5718920

Status

Public on Dec 21, 2021

Title

R.319_CD3_VDJ

Sample type

SRA

Source name

T Cells obtained from CD3

Organism

Macaca mulatta

Characteristics

sample_nhp_id: R.319
donor-nhp_id: R.319
modality: VDJ
source: CD3

Extracted molecule

total RNA

Extraction protocol

Blood and MLR Samples: RM PBMCs were isolated from whole blood by Ficoll gradient centrifugation, and then used for MLR assays either immediately, or after liquid nitrogen cryopreservation in 10% dimethyl sulfoxide (DMSO)/90% fetal bovine serum (FBS). Frozen samples were thawed using RPMI-1640 media prior to resuspension in X-vivo-15 medium (BioWhitaker) supplemented with with 10% FBS (Irvine Scientific) for MLR incubation. At the time of the MLR, stimulator PBMCs were irradiated with 3500 cGy of (137Cs) radiation. Responder PBMCs were stained with cell trace violet (CTV, Invitrogen) as per manufacturer’s instructions. 2× 105 T cell-enriched responder PBMCs along with an equal number of stimulator PBMCs were added to each well in a 96-well plate (Corning) in X-vivo-15 medium supplemented with 10% FBS and incubated at 37°C for a total of 5 days. Cell culture media change was performed on day 3 of the culture. At the end of 5 days, cells were stained with an extracellular antibody for CD3 (clone SP34-2), CD20 (clone 2H7) and CD14 (clone M5E2, all antibodies from BD Bioscience) for 20min at 4°C, and high-, medium- and non-proliferating CD3+ T cells (identified based on the dilution of CTV) were sorted and processed for single cell sequencing using the Chromium Next GEM Single Cell 5’ Reagent Kit with optimized RM primers as described below.
GvHD Organ Samples: Viable LIVE/DEAD Violet-negative CD45-positive cells were sorted from two out of four GVHD-derived samples into host and donor fractions, based on MAMU-A001 expression.
10x Genomics Single Cell 5’ Reagent Kit for Single-cell RNA-Seq and with custom RM primers for single-cell TCR-Seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Single-cell RNA - scTCR-Seq
V1_Primer_Contigs.tsv.gz

Data processing

Samples were aligned using the Cellranger multi pipeline (v6.0.2), with VDJ libraries listed as “vdj-t” to indicate T cell libraries. Samples were aggregated with the Cellranger aggr pipeline (v6.0.2) with normalize=none
We analyzed the total transcripts detected and total genes detected for each sample to determine appropriate thresholds and retained cells with 500 to 6,000 genes detected and 1,800 to 40,000 transcripts detected, and less than 5% of aligned reads originating from the mtDNA. We then applied SCTransform(52) to normalize the data, reduced the dimensionality of the dataset using PCA (retained 22 PC’s after check of ElbowPlot), clustered the data using Seurat’s FindClusters function with a resolution of 0.2, and subsetted the dataset to T cells by removing two clusters that were not enriched for the T cell genes CD3E, CD4, CD8A, or TRAC.
This resulted in a dataset with 28,646 cells by 15,904 genes. We then repeated the above procedure from SCTransform to clustering using 23 PC’s.
We then limited the dataset to samples from the PBMC, the MLR experiments, and the GVHD target organs. We then removed genes present in less than 5 cells, resulting in a dataset with 23,618 cells by 15,195 genes. This dataset was normalized using the same pipeline previously described, retaining 22 PC’s from the PCA.
Genome_build: MMul 10
Supplementary_files_format_and_content: h5 matrix of single-cell RNA-Seq

Submission date

Dec 06, 2021

Last update date

Dec 21, 2021

Contact name

Jim Kaminski

E-mail(s)

James.Kaminski@childrens.harvard.edu

Organization name

Boston Children's Hospital

Street address

1 Blackfan Circle