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Sample GSM1926584

Query DataSets for GSM1926584

Status

Public on Dec 01, 2016

Title

1772078013_E09

Sample type

SRA

Source name

hypothalamus

Organism

Mus musculus

Characteristics

strain: C57Bl6/N
age (days postnatal): 25
Sex: M
acute stress: N
cell diameter: 9.75E+00
total molecules: 9813
level1 class: oligos

Treatment protocol

Formalin stress was induced by injection of 4% PFA into the left paw

Growth protocol

C57BL/6N mice were housed conventionally (12/12-h light cycle, 55% humidity).

Extracted molecule

polyA RNA

Extraction protocol

Mice were deeply anesthetized (5% isoflurane) and transcardially perfused with 40 ml ice-cold pre-oxygenated (95% O2/5% CO2) cutting solution containing (in mM): 90 NaCl, 26 NaHCO3, 2.5 KCl, 1.2 NaH2PO4, 10 HEPES-NaOH, 5 Na-ascorbate, 5 Na-pyruvate, 0.5 CaCl2, 8 MgSO4 and 20 glucose. A central column of the mouse hypothalamus spanning the preoptic area-Arc (rostrocaudal axis), paraventricular nucleus (dorsal) and the ventrolateral hypothalamic area (lateral) was microdissected from serial 300-micrometer-thick coronal slices under microscopy guidance and dissociated using the Papain Dissociation System (Worthington). Isolated single cells were concentrated by centrifugation to a density of 600-1,000 cells/?microlitre. After mixing C1 suspension reagent (4 microlitre; "C1 reagents", Fluidigm Inc.) to the cell suspension (7 microlitre), this mixture was loaded into a C1-AutoPrep IFC microfluidic chip (Fluidigm Inc.)
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

SingleCell497

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 file of mRNA molecule counts for each gene and cell. Cells with less than 1500 molecules have been removed from this file.

Submission date

Nov 04, 2015

Last update date

May 15, 2019

Contact name

Sten Linnarsson

Organization name

Karolinska Institutet

Department

Medical Biochemistry and Biophysics

Lab

Molecular Neurobiology