GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3503590

Query DataSets for GSM3503590

Status

Public on Dec 06, 2018

Title

LS-15539_S63_E1-50

Sample type

SRA

Source name

Primary Visual Cortex (VISp)

Organism

Mus musculus

Characteristics

donor_id: 264193
donor_sex: F
donor_genotype: Rbp4-Cre_KL100/wt;Ai14(RCL-tdT)/wt
dissected_region: VISp
dissected_layer: L5
facs_gating: NeuN-positive
rna_amplification_set: A8S4_160829_01
sequencing_tube: LS-15539
sequencing_batch: RSC-041
sequencing_qc_pass_fail: Pass
cell_class: Glutamatergic
cell_cluster: cl1_e99_Gpr88_L5a.Batf3_Nuc
molecule: Nuclei

Growth protocol

All procedures were approved by the Institutional Animal Care and Use Committee at the Allen Institute for Brain Science (Protocol no. 1511). Animals were provided food and water ad libitum and were maintained on a regular 12-h day/night cycle. Mice were housed at no more than 5 adults per cage, with various enrichment materials added, including nesting materials, gnawing materials, and plastic shelters. Nutritional and foraging enrichment was provided in the form of foods such as sunflower seeds and sucrose pellets. Mice were maintained on the C57BL/6J background.

Extracted molecule

total RNA

Extraction protocol

Tissue samples were obtained from adult male and female transgenic mice carrying a Cre transgene and a Cre-reporter transgene. After euthanasia, the brain was rapidly dissected and mounted for coronal slice preparation on a Compresstome VF-300 vibrating microtome (Precisionary Instruments) and sectioned at 250 μm intervals. For whole cell dissociation, 2 mg/ml of pronase in ACSF solution was added to dissected tissues and enzymatic digestion was carried out at room temperature. After digestion,The tissue was triturated and the cell suspension was incubated on ice in preparation for fluorescence-activated cell sorting (FACS). Live cells were sorted by excluding DAPI positive events and debris, and gating to include red fluorescent events (tdTomato-positive cells). Single cells were collected into strip tubes containing 11.5μl of collection buffer (SMART-Seq v4 lysis buffer), RNase Inhibitor, and ERCCs (External RNA Controls Consortium, MIX1 at a final dilution of 1x10-8) and tubes were then stored at -80°C. For nuclei isolation, dissected tissues were placed into a homogenization buffer (10mM Tris pH 8.0, 250mM sucrose, 25mM KCl, 5mM MgCl2, 0.1% Triton-X 100, 0.5% RNase inhibitor, 1X protease inhibitor, and 0.1mM DTT). Tissues were placed into a 1ml dounce homogenizer and homogenized using 10 strokes of the loose dounce pestle followed by 10 strokes of the tight pestle to liberate nuclei . Mouse anti-NeuN antibody (EMD Millipore, MAB377, Clone A60) was added to the nuclei at a final dilution of 1:1000 and nuclei suspensions were incubated at 4°C for 30 minutes, followed by incubation in secondary antibody (goat anti-mouse IgG (H+L), Alexa Fluor 594 conjugated, ThermoFisher Scientific, 1:5000) for 30 minutes at 4°C and staining with DAPI. Single nuclei were captured by gating on DAPI-positive events, excluding debris and doublets, and then gating on Alexa Fluor 594 (NeuN) signal. Strip tubes containing FACS isolated single nuclei were then frozen at -80°C.
The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech #634894) was used per the manufacturer’s instructions for reverse transcription of single cell RNA and subsequent cDNA synthesis. Mouse whole cells were subjected to 18 PCR cycles after the reverse transcription step, whereas mouse nuclei were subjected to 21 PCR cycles. Sequencing libraries were prepared using NexteraXT (Illumina, FC-131-1096) with NexteraXT Index Kit V2 Set A (FC-131-2001). NexteraXT libraries were prepared at 0.5x volume, but otherwise followed the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

nuclei_exon_counts.csv.gz
nuclei_exon_counts.csv.gz

Data processing

50-base pair paired-end reads were aligned to GRCm38 (mm10) using a RefSeq annotation gff file retrieved from NCBI on 01/18/2016 (https://www.ncbi.nlm.nih.gov/genome/annotation_euk/all/). Sequence alignment was performed using STAR v2.5.3 (Dobin et al., 2013) in twopassMode. PCR duplicates were masked and removed using STAR option “bamRemoveDuplicates”. Only uniquely aligned reads were used for gene quantification. Gene counts were computed using the R GenomicAlignments package (Lawrence et al., 2013) sumarizeOverlaps function using “IntersectionNotEmpty”.
Reads that did not map to the genome were then aligned to synthetic constructs (i.e. ERCC) sequences and the E.coli genome (version ASM584v2). Quantification was performed using summerizeOverlaps from the R package GenomicAlignments [38].
Single nucleus quality control criteria: >500,000 genome-mapped reads, >75% reads aligned, and >50% unique reads. Single cell quality control criteria: >200,000 transcriptome mapped reads and >1000 genes detected (counts > 0).
Genome_build: mm10

Submission date

Dec 06, 2018

Last update date

Dec 09, 2018

Contact name

Allen Institute

Organization name

The Allen Institute for Brain Science

Street address

615 Westlake Ave N

City

Seattle