|
| Status |
Public on Mar 19, 2020 |
| Title |
second_experiment_D21_VDJ |
| Sample type |
SRA |
| |
|
| Source name |
T Follicular Helper (Tfh) Cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
genotype/variation: D011.10 and OTII
tissue: spleen
molecule target: VDJ RNA
|
| Growth protocol |
Single-cell suspensions were prepared from the spleens and lymph nodes of donor mice. CD4+ T cells were enriched using immunomagnetic negative selection (StemCell Technologies). For adoptive transfer experiments 0.5-1x106 CD4+ T cells were injected into recipient mice by intravenous injection. Resting B cell suspensions were cells were enriched using immunomagnetic positive selection using CD43 (StemCell Technologies).. ∼5 × 106 B1-8hi B cells (5 × 105 Igλ+, NP-specific B cells) composed of the indicated populations were injected into recipient mice by intravenous injection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For bulk RNA sequencing experiments congenic OTII Tfh cells (CD4+, CD62low, CXCR5hi, PD-1hi) or positively selected B cells (500 cells) were purified by flow cytometry 18 hours after pre-immunized host mice were injected with aDEC-205 chimeric antibodies. 1ng of total RNA was used to generate full length cDNA using Clontech’s SMART-Seq v4 Ultra Low Input RNA Kit
The cDNA was used to prepare libraries using Illumina Nextera XT DNA sample preparation kit (Cat # FC-131-1024). Libraries with unique barcodes were pooled at equal molar ratios and sequenced on an Illumina NextSeq 500 sequencer to produce 75bp reads, following manufactures protocol (Cat# 15048776). For single cell RNA sequencing, single cell spleen suspensions were prepared from half-spleens of NP-OVA immunized SellCreERT2 ROSAtdT mice on day 7 and 21 after immunization. Samples were indexed with TotalSeqC (BioLegend) cell surface antibodies and CD4+, CD62low, CD44hi, PD1hi, CXCR5high, tdTomato+ T cells were purified by flow cytometry, pooled and loaded on to a Chromium Controller (10x Genomics). Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 5′ v2 Reagent Kit (10x Genomics) according to manufacturer’s protocol. Libraries were loaded onto an Illumina NextSeq with the mid-Output Kit (150 paired end) for V-D-J analysis or NOVAseq for single cell gene expression. Hashtag indexing was used to demultiplex the sequencing data and generate gene-barcode matrices, respectively.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Library strategy: CITE-Seq
For single cell analysis we used cellranger (v3.0.2) from 10X Genomics for single-cell UMI quantification and TCR clonotype assembly. Hashtags oligos (HTOs) UMI counts were processed using CITE-Seq-Count (v1.4.0). We used Seurat (v3.1.2) [1-2], an R package to analyze single cell RNA-seq data, to identify differentially expressed genes. Genes expressed in at least 10 percent of all cells belonging to clones exhibiting expansion or contraction, with the adjusted p-value by Bonferroni correction less than 0.05 and with |average logeFC| > loge(1.1) were selected as statistical significant differentially expressed genes.
For bulk RNASeq we used kallisto (v.0.46) to map sequence reads to Mus musculus transcriptome (GRCm38/ Ensembl release 99). Kallisto TPM values were converted to absolute counts using tximport (v1.12.3) R package and DESeq2 (v.1.24.0) [3] was utilized for differential expression analysis. Differentially expressed genes were defined by having adjusted p-value < 0.05 and | logFC| > log2(1.5).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files with abundance and differential expressiong
Supplementary_files_format_and_content: HTML Report 10x Single Cell
|
| |
|
| Submission date |
Mar 18, 2020 |
| Last update date |
Mar 20, 2020 |
| Contact name |
Thiago Y Oliveira |
| E-mail(s) |
toliveira@rockefeller.edu
|
| Organization name |
The Rockefeller University
|
| Department |
Immunology, Virology and Microbiology
|
| Lab |
Laboratory of Molecular Immunology
|
| Street address |
1230 YORK AVE
|
| City |
NEW YORK |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE147182 |
Dynamic Regulation of Tfh Clonal Selection During the Germinal Center Reaction |
|
| Relations |
| BioSample |
SAMN14399334 |
| SRA |
SRX7950245 |
