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Sample GSM3142109

Query DataSets for GSM3142109

Status

Public on May 01, 2024

Title

GS1-12

Sample type

SRA

Source name

mice brain tumors

Organism

Mus musculus

Characteristics

mouse number: mouse one
strain background: Tlx-GFP;Ntv-a mice
genotype/variation: RCAS-AKT, RCAS-PDGFB and RCAS-RFP retroviruses transfection
cell types: brain tumor stem cells
sequencing type: Single-cell RNAseq

Treatment protocol

FACS Sorting.Brain tumor tissues were isolated in Phosphate buffered saline solution (PBS) and enzymatically dissociated with 0.05% Trypsin/EDTA (Gibco) in HBSS containing 2mM glucose at 37°C for 30 min. During incubation, the tissue was repeatedly triturated with a fire polished Pasteur pipette. Enzyme activity was stopped by addition of equal volume of 4% BSA in Earle’s Balanced Salt Solution (EBSS, Gibco). The cell suspension was filtered through a 70μm cell strainer and centrifuged at 1200 rpm for 5 min and the pellet was re-suspended in 0.9 M sucrose in 0.5 x HBSS (Gibco). After further centrifugation for 20 min at 2000 rpm the cell pellet was re-suspended in 2 ml of 4 % BSA in EBSS solution and placed on top of 12 ml of 4 % BSA in EBSS solution, centrifuged again for 9 min at 1500 rpm. The resulting pellet was then suspended in PBS containing Recombinant RNase Inhibitor, 1 unit/μl reaction (Clontech). After incubation with PI (1:1000) for 2 minutes; either the single cells or 1000 cells per tube were sorted at a FACS Aria (BD). Two population of cells were sorted, RFP positive and RFP / Tlx-GFP positive cells.

Growth protocol

Mice were housed according to international standard conditions and all animal experiments complied with local and international guidelines for the use of experimental animals.

Extracted molecule

polyA RNA

Extraction protocol

RNA extraction using the Arcturus PicoPure RNA Isolation kit was performed according to the manufacturer’s protocol (Life technologies). Briefly, 100 ul of extraction buffer was added to the cells, followed by brief incubation at room temperatue. While the lysate was incubated, the purification column was wetted (Pre-condition) by adding 250 ul of condition buffer, followed by centrifugation at 16,000 × g for 1 min. 100 ul of 70% ethanol was then added to the lysate, mixed well and the whole mixture was transferred to the prepared purification columns. Purification columns were centrifuged for 2 min at 100 × g, followed by centrifuging at 16,000 × g for 30 sec to remove flow through. 100 ul of wash buffer 1 was added to the purification columns, which were then centrifuged for 1 min at 8,000 × g. DNase treatment may was performed directly within the purification column. 10 µl DNase I stock solution (Qiagen, catalog#79254) was mixed with 30 µL Buffer RDD. 40 µL DNase incubation mix was applied directly into the purification column membrane followed by Incubation at room temperature for 15 minutes. 40 ul of wash buffer 1 was added to the purification columns, which were then centrifuged for 15 seconds at 8,000 × g. After that, 100 ul of wash buffer 2 was added to the purification columns, which were then centrifuged for 1 min at 8,000 × g. Another 100 ul of wash buffer 2 was added to the purification columns. Purification columns were centrifuged at 16,000 × g for 2 min. Another spin at 16,000 × g for 1 min was performed to remove the wash residue in the columns. Purification columns were transferred to new Eppendorf tubes and 11 ul of elution buffer was carefully added onto the column membrane followed by 1 min incubation at room temperature. Purification Columns were centrifuged at 1,000 × g for 1 min, and subsequently at 16,000 × g for 1 min to elute RNA. 10 ul of RNA solution was obtained.
Libraries were constructed using the Nextera XT kit as recommended by the manufacturer (Illumina, San Diego, USA). 0.5 ng of cDNA was tagmented, utilizing 5 μl of Amplicon Tagment Mix with 10 μl Tagment DNA Buffer. Tagmentation reaction was performed by incubation for 20 min at 55°C. Next, index primers were added together with Nextera PCR Master Mix. Tagmented cDNA was amplified by limited-cycle PCR (10 cycles). Libraries were purified using AMPure beads (Beckman Coulter, High Wycombe, UK) and quality validated using Agilent 2100 BioAnalyzer. Libraries were normalized and 8 nM pooled libraries were then sequenced on Illumina HiSeq2000.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

AS-169619-LR-26120

Data processing

RTA 1.18.64 was used for base-calling
For all single-cell/bulk libraries from neural stem cells or brain tumours, we obtained the gene expression profiles via mapping the sequencing reads to mouse mm10 transcriptome by RSEM (v1.3.0) (Li and Dewey, 2011). For single cells from brain tumours, we kept cells with 200 to 3500 genes and less than 10% of reads on mitochondrial genes and projected the cells into low dimension space using Monocle (Qiu et al., 2017; Trapnell et al., 2014) in R (R Core Team, 2018). We calculated the spearman correlation coefficient for single cells in neural stem cell populations and brain tumour populations using log2 transformed TPM value.
We used the R package DESeq2 (Love et al., 2014) to estimate the differentially expressed genes between bulk Tlx-GFP+/dsRed+ and Tlx-GFP-/dsRed+ using the estimated counts from RSEM results. The statistcally signficant genes were selected with fdr <= 0.1. Heatmaps for selected genes were produced with ComplexHeatmap (Gu et al., 2016).
Genome_build: mm10
Supplementary_files_format_and_content: csv

Submission date

May 15, 2018

Last update date

May 01, 2024

Contact name

Haikun Liu

E-mail(s)

l.haikun@dkfz.de

Organization name

DKFZ

Street address

INF 581