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Sample GSM1850773

Query DataSets for GSM1850773

Status

Public on Apr 05, 2016

Title

Cy72_CD45_H03_S759_comb

Sample type

SRA

Source name

melanoma tumor

Organism

Homo sapiens

Characteristics

tumor-of-origin: Cy72

Treatment protocol

Resected tumors were transported in DMEM (ThermoFisher Scientific) on ice immediately after surgical procurement. Tumors were rinsed with PBS (Life Technologies). A small fragment was stored in RNA-protect (Qiagen) for bulk RNA and DNA isolation. Using scalpels, the remainder of the tumor was minced into tiny cubes <1 mm3 and transferred into a 50 ml conical tube (BD Falcon) containing 10 ml pre-warmed M199-media (ThermoFisher Scientific), 2 mg/ml collagenase P (Roche) and 10U/μl DNase I (Roche). Tumor pieces were digested in this digestion media for 10 minutes at 37°C, then vortexed for 10 seconds and pipetted up and down for 1 minute using pipettes of descending sizes (25 ml, 10 ml and 5 ml). If needed, this was repeated twice more until a single-cell suspension was obtained. This suspension was then filtered using a 70μm mesh (ThermoFisher Scientific) and residual cell clumps were discarded. The suspension was supplemented with 30 ml PBS (Life Technologies) with 2% fetal calf serum (FCS) (Gemini Bioproducts) and immediately placed on ice. After centrifuging at 580g at 4°C for 6 minutes, the supernatant was discarded and the cell pellet was re-suspended in PBS with FCS and placed on ice prior to staining for FACS. Single-cell suspensions were stained with CD45-FITC (VWR), Calcein-AM (Life Technologies) and CD90-PE (BioLegend) per manufacturer recommendations. First, doublets were excluded based on forward and sideward scatter, then we gated on viable cells (Calceinhigh) and sorted single cells (CD45+ or CD45-CD90+ or CD45-CD90-) into 96-well plates chilled to 4°C, pre-prepared with 10μl TCL buffer (Qiagen) supplemented with 1% beta-mercaptoethanol (lysis buffer). Single-cell lysates were vortexed, spun down at 3700 rpm at 4°C for 2 minutes, immediately placed on dry ice and transferred for storage at -80°C.

Growth protocol

no culturing (freshly-resected tumor samples)

Extracted molecule

polyA RNA

Extraction protocol

RNA and DNA were isolated using the Qiagen minikit following the manufacturers recommendations. Whole Transcriptome amplification (WTA) was performed with a modified SMART-Seq2 protocol, with Maxima Reverse Transcriptase (Life Technologies) used in place of superscript II.
WTA products were cleaned with Agencourt XP DNA beads and 70% ethanol (Beckman Coulter) and Illumina sequencing libraries were prepared using Nextera XT (Illumina), as previously described (15). The 96 samples of a multiwall plate were pooled together, and cleaned with two 0.8x DNA SPRIs (Beckman Coulter). Library quality was assessed with a high sensitivity DNA chip (Agilent) and quantified with a high sensitivity dsDNA Quant Kit (Life Technologies). Samples were sequenced on an Illumina NextSeq 500 instrument using 30bp paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

single cell RNA-seq

Data processing

Following sequencing on the NextSeq, BAM files were converted to merged, demultiplexed FASTQs. Paired-end reads were then 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", which allows alignment of sequences with single base changes such as due to point mutations. Expression levels of genes were quantified as Ei,j=log2(TPMi,j/10+1), where TPMi,j refers to transcript-per-million (TPM) for gene i in sample j, as calculated by RSEM v1.2.3 in paired-end mode. TPM values were divided by 10 since we estimate the complexity of our single cell libraries to be on the order of 100,000 transcripts and would like to avoid counting each transcript ~10 times, as would be the case with TPM, which may inflate the difference between the expression level of a gene in cells in which the gene is detected and those in which it is not detected. For each cell, we quantified 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 excluded all cells with either fewer than 1,700 detected genes or an average housekeeping expression (E, as defined above) below 3. Only genes detected with E>2 in at least 5 cells were retained in the processed data file.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text file containing the normalized expression levels (E) for all genes with E>2 in at least 5 cells, across 3,249 cells that passed QC. the tumor-of-origin is indicated in the name of each cell (CY##). The first row denotes the classification (based on inferred CNVs) to malignant (2), non-malignant (1) or unresolved (0) cells. The second row denotes the inferred cell types for non-malignant cells, including T-cells (1), B-cells (2), Macrophages (3), Endothelial cells (4) and CAFs (5). All other rows correspond to genes.

Submission date

Aug 13, 2015

Last update date

Nov 07, 2019

Contact name

Itay Tirosh

E-mail(s)

Tirosh.itay@gmail.com

Organization name

WEIZMANN INSTITUTE OF SCIENCE

Street address

Herzl 234