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Sample GSM1839577

Query DataSets for GSM1839577

Status

Public on Jan 04, 2016

Title

Gad2_tdTpositive_cell_68

Sample type

SRA

Source name

Primary visual cortex

Organism

Mus musculus

Characteristics

mouse line abbreviation: Gad2
driver 1: Gad2-IRES-Cre
reporter: RCL-tdT (Ai14)
brain region: VISp
dissection: All
input material: single cell
tdtomato labelling: positive
passes qc checks: Y
primary type: Sst Cbln4
allen science vignette cell id: D1352_V

Growth protocol

Animals were provided food and water ad libitum and were maintained at a regular 12 h day/night cycle at no more than 5 adult animals per cage. Animals were maintained on the C57BL/6J background.

Extracted molecule

total RNA

Extraction protocol

Individual adult male mice (P56±3) were anesthetized in an isoflurane chamber, decapitated, and the brain was immediately removed and submerged in fresh ice-cold artificial cerebrospinal fluid (ACSF). The brain was sectioned on a vibratome (Leica VT1000S) on ice, and each slice (300-400 µm) was immediately transferred to an ACSF bath at room temperature. After the brain slicing is complete (not more than 15 minutes), individual slices of interest were transferred to a small Petri dish containing bubbled room temperature ACSF. The regions of interest (all layers of VISp or specific layers of VISp) were microdissected under a fluorescence dissecting microscope, and the slices before and after dissection were imaged to later examine the location of the microdissected tissue and confirm its location within V1. The dissected tissue pieces were transferred to a microcentrifuge tube and treated with 1 mg/ml pronase in carbogen-bubbled ACSF for 70 minutes at room temperature without mixing in a closed tube. After incubation, with the tissue pieces sitting at the bottom of the tube, the pronase solution was pipetted out of the tube and exchanged with cold ACSF containing 1% fetal bovine serum. The tissue pieces were dissociated into single cells by gentle trituration through Pasteur pipettes with polished tips of 600, 300, and 150-µm diameter. Single cells were isolated by FACS into individual wells of 96-well plates or 8-well PCR strips containing 2.275 µl of Dilution Buffer (SMARTer Ultra Low RNA Kit for Illumina Sequencing, Clontech Cat#634936), 0.125 µl RNase inhibitor (SMARTer kit), and 0.1 µl of 1:1,000,000 diluted RNA spike-in RNAs (ERCC RNA Spike-In Mix 1, Life Technologies Cat#4456740). Sorting was performed on a BD FACSAriaII SORP using a 130um nozzle, a sheath pressure of 10psi, and in the single cell sorting mode. To exclude dead cells, DAPI (DAPI*2HCl, Life Technologies Cat#D1306) was added to the single cell suspension to the final concentration of 2 ng/ml. FACS populations were chosen to select cells with low DAPI and high tdTomato (tdT) fluorescence. In some cases, we also selected cells that have low DAPI and low tdT fluorescence, in order to capture tdT-negative cells from a sample. To collect all cells in an unbiased manner, we selected all cells with low DAPI fluorescence, regardless of their tdT fluorescence level. Sorted cells were frozen immediately on dry ice and stored at -80°C.
We used the SMARTer kit from Clontech (SMARTer Ultra Low RNA Kit for Illumina Sequencing, Clontech Cat#634936) to reverse transcribe single cell RNA, and amplify the cDNA for 19 PCR cycles. To stabilize the RNA after quickly thawing the cells on ice, we immediately added to each sample an additional 0.125 µl of RNase inhibitor mixed with SMART CDS Primer II A. All steps downstream were carried out according to the manufacturer’s instructions. Each amplification experiment included a set of controls: 10 pg cortex RNA (isolated from Rbp4-Cre;Ai14, P57 male) as positive control for amplification, ERCC-only control to demonstrate the absence of RNases throughout the sorting process, and water-only control, to control for specificity of amplification/absence of contamination. cDNA concentration was quantified using Agilent Bioanalyzer High Sensitivity DNA chips. For most samples, 1 ng of amplified cDNA was used as input to make sequencing libraries with Nextera XT DNA kit (Illumina Cat#FC-131-1096). For smaller cells (e.g., glia), which did not consistently produce more than 1ng cDNA, we used 0.5–1 ng cDNA as input. We stopped the procedure after PCR clean-up and did not perform library normalization or library pooling. Individual libraries were quantified using Agilent Bioanalyzer DNA 7500 chips. In order to assess sample quality and adjust the concentrations of libraries for multiplexing on HiSeq, all libraries were sequenced first on Illumina MiSeq to obtain approximately 100,000 reads per library, and then on Illumina HiSeq 2000 or 2500 to generate 100 bp reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

D10011431

Data processing

Adapter sequences were clipped from 100 base-pair single-end reads in FASTQ format using the ea-utils application fastq-mcf with settings -C 200000 to filter against Nextera adapter sequences.
Trimmed reads were aligned to GRCm38 (mm10) using the RefSeq annotation gff file downloaded on 6/1/2013. Transcriptome alignment files in BAM format were generated with RSEM v1.2.4 and Bowtie v1.1.0 using the program rsem-calculate-expression with settings --keep-intermediate-files --bowtie-chunkmbs 2000 --output-genome-bam --bowtie-e 500 --bowtie-m 100.
Reads that did not map to the RefSeq annotations were aligned to ERCC sequences and tdTomato sequence using Bowtie v1.1.0 with settings -S --chunkmbs 2000.
Genome_build: mm10
Supplementary_files_format_and_content: CSV: Comma-separated tables of data values; Columns are samples and Rows are genes or ERCC/tdTomato. Column names correspond to sample Titles; Row names correspond to RefSeq Gene Symbols, ERCC symbols, or tdTomato.

Submission date

Jul 31, 2015

Last update date

May 15, 2019

Contact name

Lucas T Gray

E-mail(s)

lucasg@alleninstitute.org

Organization name

Allen Institute for Brain Science

Department

Molecular Genetics