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Sample GSM2760793

Query DataSets for GSM2760793

Status

Public on Nov 30, 2017

Title

HN26_P14_B11_S215_comb

Sample type

SRA

Source name

Head and neck cancer single cell

Organism

Homo sapiens

Characteristics

enzyme used: Maxima
tumor site: Lymph node

Treatment protocol

Tumor Dissociation: Fresh biopsy samples of oral cavity HNSCC were minced, washed with phosphate buffered saline (PBS; ThermoFisher Scientific, Waltham, MA), and dissociated using a Human Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) per manufacturer guidelines. Viability was confirmed to be >90% in all samples using trypan blue (ThermoFisher Scientific) exclusion. Cell suspensions were filtered using a 70 ?m filter (ThermoFisher Scientific), and dissociated cells were pelleted and re-suspended in PBS with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO). Cells were stained with CD45-vioblue (Miltenyi Biotec), along with either the combination of CD90-PE (BD Biosciences, Franklin Lakes, NJ) and CD31-PE-cy7 (BD Biosciences) or CD3-PE-cy7 (ThermoFisher Scientific), then washed with cold PBS, and re-suspended for flow cytometry analyses. Sorting of Patient Samples: Cells were stained for viability with 1 ?M calcein AM (ThermoFisher Scientific) and 0.33 ?M TO-PRO-3 iodide (ThermoFisher Scientific) immediately prior to sorting. Fluorescence-activated cell sorting (FACS) was performed on FACSAria Fusion Special Order System (BD Biosciences) using 488 nm (calcein AM, 530/30 filter), 640 nm (TO-PRO-3, 670/14 filter), 405 nm (Vioblue, 450/50 filter), 561 nm (PE, 586/15 filter; PE-Cy7, 780/60 filter) lasers. Standard forward scatter height versus area criteria were used to discard doublets and capture singlets. Viable cells 45 were identified as calceinhigh and TO-PROlow and additional gates were used to enrich or deplete specific cell types in each plate. For each tumor, plates were sorted containing CD45- cells (to deplete immune cells), CD45-/CD90-/CD31- cells (to further deplete fibroblasts and endothelium and enrich for malignant cells), CD45+ cells (to enrich for immune cells), and CD45+/CD3+ cells (to enrich specifically for T-cells). Single cells were sorted into 96-well plates containing TCL buffer (Qiagen, Hilden, Germany) with 1% ?-mercaptoethanol. Plates were briefly centrifuged, snap frozen, and stored at ?80 °C before cDNA synthesis and library construction. For each tumor sample, at least one CD45- and one CD45+ plate was sequenced.

Extracted molecule

polyA RNA

Extraction protocol

RNA was purified using Agencourt RNAClean XP beads (Beckman Coulter, Brea, CA),
Libraries for isolated single cells were generated based on the SMART-Seq2 protocol (Picelli et al., 2013; Picelli et al., 2014) with the following modifications:purified RNA was subjected to reverse transcription with Superscript II (ThermoFisher Scientific) or Maxima (ThermoFisher Scientific) reverse transcriptase and whole transcriptome amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA). Full length cDNA libraries were tagmented using the Nextera XT Library Prep Kit (Illumina, San Diego, CA). 384 samples were pooled and sequenced as paired-end 38 base reads on a NextSeq 500 instrument (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Paired-end, 38-base reads were mapped to the UCSC hg19 human transcriptome using Bowtie with parameters "-q --phred33-quals -n 1 -e 99999999 -l 25 -I 1 -X 2000 -a -m 15 -S -p 6". Expression values were calculated by RSEM v1.2.3 in paired-end mode using parameters "--estimate-rspd --paired end -sam -p 6", from which TPM values for each gene were extracted.
For each cell, we quantified two quality measures: the number of genes for which at least one read was mapped, and the average expression level of a curated list of housekeeping genes. We then conservatively excluded all cells with either fewer than 2,000 detected genes or an average housekeeping expression (E, as defined above) below 2.5.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file containing TPM values ( transcript-per-million reads) for 23,686 analyzed genes (rows), across 5902 cells (columns, sample names indicated at the top row). Headers indicate the enzyme being used for reverse transcription, the sample site (Primary vs. lymph node), the classification into cancer and non-cancer cells and the inferred non-cancer cell types.

Submission date

Aug 30, 2017

Last update date

Nov 30, 2017

Contact name

Itay Tirosh

E-mail(s)

Tirosh.itay@gmail.com

Organization name

WEIZMANN INSTITUTE OF SCIENCE

Street address

Herzl 234