GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2377986

Query DataSets for GSM2377986

Status

Public on Mar 31, 2017

Title

MGH42_P14_E08

Sample type

SRA

Source name

astrocytoma single cell

Organism

Homo sapiens

Characteristics

tumor: MGH42

Treatment protocol

Fresh tumor tissue was minced with a scalpel and enzymatically dissociated using a gentle papain-based brain tumor dissociation kit (Miltenyi Biotec). Large pieces of debris were removed with a 100 micron strainer, and dissociated cells were layered carefully onto a 5mL density gradient (Lympholyte-H, Cedar Lane labs), which was centrifuged at 2,000 rpm for 10 min at room temperature to pellet dead cells and red blood cells. The interface containing live cells was saved and used for staining and flow cytometry. Tumor cells were blocked in 1% bovine serum albumin in Hanks buffered saline solution (BSA / HBSS), and then stained first with CD45-Vioblue direct antibody conjugate (Miltenyi Biotec) for 30 min at 4C. Cells were washed with cold PBS, and then resuspended in 1 mL of BSA / HBSS containing 1uM calcein AM (Life Technologies) and 0.33uM TO-PRO-3 iodide (Life Technologies) to co-stain for 30 min before sorting. Fluorescence-activated cell sorting was performed on FACSAria Fusion Special Order System (Becton Dickinson) using 488nm (calcein AM, 530/30 filter), 640nm (TO-PRO-3, 670/14 filter), and 405nm (Vioblue, 450/50 filter) lasers. Fluorescence-minus-one controls were included with all tumors, as well as heat killed controls in early pilot experiments, which were crucial to ensure proper identification of the TO-PRO-3 positive compartment and ensure sorting of the live cell population. Standard, strict forward scatter height versus area criteria were used to discriminate doublets and gate only singlets. Viable cells were identified by staining positive with calcein AM but negative for TO-PRO-3. Single cells were sorted into 96-well plates containing cold buffer TCL buffer (Qiagen) containing 1% beta-mercaptoethanol, snap frozen on dry ice, and then stored at -80C prior to whole transcriptome amplification, library preparation and sequencing.

Growth protocol

no culutring (freshly-resected tumor samples)

Extracted molecule

polyA RNA

Extraction protocol

RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter)
Libraries from isolated single cells were generated based on the Smart-seq2 protocol (Picelli 2014) with the following modifications.oligo-dT were used to prime reverse transcription with Maxima reverse transcriptase and locked TSO oligonucleotide, which was followed by 20 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) with subsequent Agencourt AMPure XP bead purification as described. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina) with custom barcode adapters (sequences available upon request). Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina).
single cell RNA-seq

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", which allows alignment of sequences with single base changes such as point mutation in the IDH1 gene. Expression values were calculated from SAM files using 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.
Expression levels were quantified as Ei,j=log2(TPMi,j/10+1), where TPMi,j refers to transcriptper-million for gene i in sample j, as calculated by RSEM. TPM values are divided by 10 since we estimate the complexity of single cell libraries in 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 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 3,000 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 the normalized expression levels (E) for 23,686 analyzed genes (rows, first, column indicates Gene Symbols) across 6341 astrocytoma cells (columns, sample names indicated at the top row( that passed QC from 10 tumors.

Submission date

Nov 04, 2016

Last update date

Mar 31, 2017

Contact name

Itay Tirosh

E-mail(s)

Tirosh.itay@gmail.com

Organization name

WEIZMANN INSTITUTE OF SCIENCE

Street address

Herzl 234