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Sample GSM7567335

Query DataSets for GSM7567335

Status

Public on Apr 10, 2024

Title

HTO_CD3KO_MR1KO_chimera

Sample type

SRA

Source name

Thymus

Organism

Mus musculus

Characteristics

tissue: Thymus
cell type: MAIT

Extracted molecule

other

Extraction protocol

Single-cell suspension of thymus was obtained as described (Salou et al. 2019). MR1:5-OP-RU tetramer staining along with an anti-TCRb-APC (clone H57-597; 1:200) was performed at room temperature for 30min in PBS 0.5% BSA 2 mM EDTA complemented with rat anti-mouse CD16/CD32 antibody and MR1:6FP to block non-specific binding following the National Institutes of Health (NIH) Tetramer Core Facility instructions. Tetramer-based magnetic enrichment was performed as described (F. P. Legoux and Moon 2012). Cells were washed and stained for surface markers with following monoclonal antibodies: anti-CD44-APC-Cy7 (IM7, Biolegend; 1:400), -CD24-PE-Cy5 (M1/69, Biolegend; 1:1600), -CD19-AF700 (ID3, Biolegend; 1:800), -B220-AF700 (RA3-6B2, eBioscience; 1:400), -CD11c-AF700 (N418, eBioscience; 1:200), -F4/80-AF700 (BM8, Biolegend; 1:200). For cell hashing in combination with 10x Chromium Single Cell 5′ Solution, TotalSeq-C anti-mouse Hashtags 2 to 7 (M1/42; 39-F11, Biolegend, 155863, 155865, 155867, 155869, 155871, 155873; 1:100 for all) were used according to manufacturer recommendations. Single-cell suspensions from 6 hematopoietic chimeras thymi were pooled and DAPI (1:1000) staining was added just before FACS sorting on an Aria III cell sorter (BD). Total TCRb+MR1:5-OP-RU+ cells were flow-sorted into PBS 0.35% BSA, centrifuged, counted, and 6,500 cells were loaded onto the Chromium 5′ chip. Reverse transcription, library preparation and sequencing were performed according to manufacturer recommendations (10× Genomics).
Three libraries were performed according to the manufacter’s instructions (single cell 5’ with feature barcode v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). Total RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.
5' VDJ scRNA-seq
CITE-seq

Library strategy

OTHER

Library source

other

Library selection

other

Instrument model

Illumina MiSeq

Description

10x Genomics

Data processing

The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v3 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger (version 6.0.0)
Assembly: mm10-2020-A for the scRNA-seq and vdj_GRCm38_alts_ensembl-3.1.0 for scTCR-seq

Submission date

Jul 06, 2023

Last update date

Apr 10, 2024

Contact name

Anne-laure Le gac

E-mail(s)

annelaure.legac@gmail.com

Phone

0630777988

Organization name

Institut Curie