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Sample GSM3137136

Query DataSets for GSM3137136

Status

Public on Feb 15, 2019

Title

N722_S503_3: CD69neg scRNA-seq

Sample type

SRA

Source name

CD69- CD8+ T cells

Organism

Homo sapiens

Characteristics

passages: FACS purified cells
tissue: mesenteric LN
cell type: CD69- CD8+ T cells

Extracted molecule

total RNA

Extraction protocol

scRNA-seq: Single-cell analysis was derived from a previously published method (Shalek et al., Nature, 2014). Single HIV-tetramer+ cells were index sorted using a FACSAria II (BD Biosciences) directly into 96-well microtiter plates containing lysis buffer. In total, 552 cells were sorted. Cellular nucleic acids were recovered by processing the lysates with an RNEasy Plus Micro Kit (Qiagen) and then binding to RNAClean (SPRI) beads (Beckman Coulter). ERCC RNA control (Ambion) was diluted 1 in 107 from stock, and 10% by volume was added to the RNAClean beads prior to purification.
scRNA-seq: Libraries were prepared by getting mRNA be reverse transcribed from an anchored oligo-dT primer that includes a universal priming site at the 5’ end. RT reactions also included a template-switch oligonucleotide to allow the incorporation of the universal priming site at cDNA 3’ ends. Whole transcriptome cDNA was then amplified by PCR for 22–24 cycles using universal priming sites primers and Kapa HiFi Hot Start ReadyMix. After post-PCR clean-up, amplified whole transcriptome cDNA was incorporated into barcoded Illumina Nextera libraries and sequenced to a depth of about 2 million 150bp paired-end reads/cell on a HiSeq 4000 (Illumina). and amplified for 14 PCR cycles. Libraries were pooled and paired-end sequenced (38bp+37bp).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

scRNA-seq: Data were initially filtered for quality. Paired-end sequencing reads were then trimmed to remove adaptor sequences and low-quality basecalls using the utility Trimmomatic. Reads were subsequently aligned using Bowtie against a genomic repeat element database derived from RepeatMasker software to quantify and remove transcript reads considered to be irrelevant for the analysis in hand (rRNA, rRNA, LINEs, SINEs, etc). The remaining reads were aligned against the most recent human genome build, hg38, using the splice-site aware aligner, STAR. As a bioinformatic measure of library quality, the total frequency of reads that could be mapped to the transcriptome was determined, and outliers were excluded from further analysis. Transcripts were then quantified and normalized using the bioinformatics package, Cufflinks. In our experience, a small number of libraries display low complexity and lack expression of highly expressed housekeeping genes. In most systems, a set of housekeeping genes should be expressed in every cell. We therefore use qPCR to identify viable samples expressing two distinct housekeeping genes. Samples with low or no expression of these genes are removed from Nextera library construction. To further ensure exclusion of data from single cells for which library construction was unsuccessful, we determined diversity (Shannon entropy and count of detected genes) using the R package, Vegan, and discarded outliers.
Analysis of TCR sequences were conducted through the software package, MiXCR, to assemble and annotate TCR sequences from the pool of reads which had not aligned to the reference genome in the previous step. After assembly, low quality and low frequency TCR chains were removed. Clonotypes were then built for each cell using the refined chain information. An average of 55 (range 23–143) TCRBV sequences were analyzed per patient.
Genome_build: mm10
Supplementary_files_format_and_content: EC_CD69_count.csv

Submission date

May 08, 2018

Last update date

Nov 14, 2019

Contact name

Marcus Buggert

E-mail(s)

marcus.buggert@ki.se

Organization name

Karolinska Institutet

Department

Microbiology