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Sample GSM2160607

Query DataSets for GSM2160607

Status

Public on Jun 10, 2016

Title

C1-1772060-224-C04

Sample type

SRA

Source name

cortex S1

Organism

Mus musculus

Characteristics

strain: CD1
age: p24
Sex: F
treatment: No
inferred cell type: MOL1

Treatment protocol

In the stress treatment mice, acute stress was induced injection of 4% PFA into the left paw.

Growth protocol

Wild-type (CD1 or C57BL6) and transgenic mice (5Pdgfratm11(EGFP) Sor/J, Lhx6 Cre RFP and PDGFR-GFP) between post-natal day 21 and 31 of both sexes were used. In the PDGFRa-GFP knock-in mice, H2B-eGFP fusion gene is expressed under the promoter of the oligodendrocyte precursor marker PDGFRa. The Lhx6–Cre transgenic mouse is a BAC transgenic mouse that expresses Cre under transcriptional control of Lhx6. This mouse line was crossed with the Cre-reporter line where tandem-dimer red fluorescent protein (tdRFP) is ubiquitously expressed under the ROSA26 locus. In the BAC transgenic mouse 5HT3aEGFP, EGFP is expressed under control of the Htr3a promoter.

Extracted molecule

polyA RNA

Extraction protocol

Selected CNS regions were dissected in 300µm thick vibratome sections and cut out with scalpel. Tissue was then dissociated into a single cell suspension (described in Zeisel & Munoz-Manchado, Science 2015 Mar 6;347(6226):1138). For the spinal cord, an extra gradient step was used after the strainer and final centrifugation step. The pellet was ressuspended in 2ml Neurobasal-A medium including supplements and transferred onto an Optiprep Density Gradient, which consisted of 3 phases (Bottom phase: 160µl Optiprep + 340µl Neurobasal-A; middle phase: 220µl Optiprep + 1780µl Neurobasal-A; top phase: 75µl Optiprep + 925µl Neurobasal-A). The gradient was then centrifuged at 300g for 10min at 8°C and cells were located between the bottom and the middle phase. Cell suspension in a concentration of 600-1000 cells/μL was used. C1 Suspension Reagent was added in a ratio of 4 μL to every 7 μL cell suspension. 11 μL of the cell suspension mix was loaded on a C1 Single-Cell AutoPrep IFC microfluidic chip designed for 10- to 17-μm cells, and the chip was then processed on a Fluidigm C1 instrument using the 'mRNA Seq: Cell Load (1772x/1773x)' script (30 min at 4°C).
Lysis mix, RT mix and PCR mix (described in Islam et al., Nat Methods. 2014 Feb;11(2):163-6) were added to the chip. The plate was placed in the Fluidigm C1 instrument and the 'mRNA Seq: RT + Amp (1772x/1773x)' script was executed, and included lysis, reverse transcription and 21 cycles of PCR. The amplified cDNA was harvested in a total of 13 μL C1 Harvesting Reagent. Amplified cDNA was simultaneously fragmented and barcoded by tagmentation using Tn5 DNA transposase to transfer adaptors to the target DNA as described (Ibid.). 100 µl Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were washed in 2× BWT (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) and resuspended in 2 ml 2× BWT. Twenty microliters of beads were added to each well and incubated at room temperature for 15 min. All fractions were pooled, the beads were immobilized and the supernatant removed. The beads were resuspended in 100 μL TNT (20 mM Tris, pH 7.5, 50 mM NaCl, 0.02% Tween), washed in 100 μL Qiagen Qiaquick PB, and twice in 100 μL TNT. The beads were resuspended in 100 μL restriction mix (1× NEB CutSmart, 0.4 U/μL PvuI-HF enzyme), designed to cleave 3′ fragments carrying a PvuI recognition site. The mix was incubated for 1 h at 37 °C, then washed three times in TNT. Finally, to elute DNA, beads were resuspend in 30 µL ddH2O and incubated 10 minutes at 70°C. Beads were then immediately bound to magnet and the supernatant was collected. To remove short fragments, Ampure beads (Beckman Coulter) were used at 1.8× volume and eluted in 30 µL.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Marques_et_al_mol_counts2.tab.gz

Data processing

Read processing was performed as described (Islam et al., Nat Methods. 2014 Feb;11(2):163-6), except that we removed any RNA molecule (i.e. Unique Molecular Identifier) supported only by a single read ("singleton molecules"). This removed a large number of false positive molecules, artefacts that can arise by sequencing error, PCR-induced mutations or translocations and cross-contamination.
The first 6 bases of each read represent the random Unique Molecular Identifier used for molecule counting. After follows three or more Gs, stemming from the template switching at the mRNA 5' end during first strand cDNA sythesis.
Genome_build: UCSC mm10
Supplementary_files_format_and_content: Tab-delimited table of total number of detected mRNA molecules from each gene in each cell

Submission date

May 19, 2016

Last update date

May 15, 2019

Contact name

Sten Linnarsson

Organization name

Karolinska Institutet

Department

Medical Biochemistry and Biophysics

Lab

Molecular Neurobiology

Street address

Scheeles väg 1

City

Stockholm

ZIP/Postal code

171 65

Country

Sweden

Platform ID

GPL13112

Series (1)

GSE75330

RNA-seq analysis of single cells of the oligodendrocyte lineage from nine distinct regions of the anterior-posterior and dorsal-ventral axis of the mouse juvenile central nervous system

Relations

BioSample

SAMN05077153

SRA

SRX1776735

Supplementary data files not provided

SRA Run Selector

Raw data are available in SRA

Processed data are available on Series record