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Sample GSM3189916 Query DataSets for GSM3189916
Status Public on Oct 31, 2018
Title VISp_Ctgf-T2A-dgCre/wt;Snap25-LSL-F2A-GFP/wt_No Injection_1
Sample type SRA
 
Source name VISp_Ctgf-T2A-dgCre/wt;Snap25-LSL-F2A-GFP/wt_No Injection
Organism Mus musculus
Characteristics donor_genotype: Ctgf-T2A-dgCre/wt;Snap25-LSL-F2A-GFP/wt
injection_type: No Injection
tissue: Primary Visual Cortex (VISp)
Treatment protocol For retrogradely-labeled samples, we injected AAV2-retro-EF1a-Cre (Tervo et al., 2016), SADB-rabies-dGdL-Cre, or CAV-Cre (gift of Miguel Chillon Rodrigues, Universitat Autònoma de Barcelona) (Hnasko et al., 2006) into brains of heterozygous or homozygous Ai14 mice as previously described (Tasic et al., 2016). For ALM experiments, we also injected AAV2-retro-CAG-GFP or AAV2-retro-CAG-tdTomato (Tervo et al., 2016) into wild-type mice. For two VISp experiments, we injected into SCs by inserting the needle through the cerebellum at a 45°-angle in the posterior to anterior direction. TdT+ or GFP+ single cells were isolated from VISp or ALM, depending on the injection area.
Growth protocol All procedures were carried out in accordance with Institutional Animal Care and Use Committee protocols 1508, 1510, and 1511 at the Allen Institute for Brain Science. Animals were provided food and water ad libitum and were maintained on a regular 12-h day/night cycle at no more than five adult animals per cage. Animals were maintained on the C57BL/6J background, and newly received or generated transgenic lines were backcrossed to C57BL/6J. Experimental animals were heterozygous for the recombinase transgenes and the reporter transgenes. Standard tamoxifen treatment for CreER lines included a single dose of tamoxifen (40 μl of 50 mg ml−1) dissolved in corn oil and administered via oral gavage at postnatal day (P)10–14. Tamoxifen treatment for Nkx2.1-CreERT2;Ai14 was performed at embryonic day (E)17 (oral gavage of the dam at 1 mg per 10 g of body weight), pups were delivered by cesarean section at E19 and then fostered. Cux2-CreERT2;Ai14 mice received tamoxifen treatment at P35 ± 5 for five consecutive days. Trimethoprim was administered to animals containing Ctgf-2A-dgCre by oral gavage at P40 ± 5 for three consecutive days (0.015 ml per g of body weight using 20 mg ml−1 trimethoprim solution). Ndnf-IRES2-dgCre animals did not receive trimethoprim induction, since the baseline dgCre activity (without trimethoprim) was sufficient to label the cells with the Ai14 reporter (Tasic et al., 2016). We excluded any animals with anophthalmia or microphthalmia.
Extracted molecule total RNA
Extraction protocol We isolated single cells as previously described (Hempel et al., 2007; Sugino et al., 2006; Tasic et al., 2016) with modifications. We mostly employed layer-enriching dissections, with focus on a single layer. Broader dissections (no layer enrichment or multiple layers combined) were employed for lines which label small numbers of cells, in order to facilitate isolation of sufficient number of cells. We updated our ACSF formulation to consist of CaCl2 (0.5 mM), glucose (25 mM), HCl (96 mM), HEPES (20 mM), MgSO4 (10 mM), NaH2PO4 (1.25 mM), myo-inositol (3 mM), N-acetylcysteine (12 mM), NMDG (96 mM), KCl (2.5 mM), NaHCO3 (25 mM), sodium L-ascorbate (5 mM), sodium pyruvate (3 mM), taurine (0.01 mM), thiourea (2 mM), and bubbled with carbogen gas (95% O2 and 5% CO2). For samples collected after 12/16/2016, the ACSF formulation also included trehalose (13.2 mM). Mice were anesthetized with isoflurane and perfused with cold carbogen-bubbled ACSF. The brain was dissected, submerged in ACSF, embedded in 2% agarose, and sliced into 250-µm coronal sections on a compresstome (Precisionary). Enzymatic digestion, trituration into single cell suspension, and fluorescence-activated cell sorting (FACS) of single cells were carried out as previously described (Tasic et al., 2016). Cells were sorted into 8-well strips containing lysis buffer from SMART-Seq v4 kit with RNase inhibitor (0.17 U/µl), immediately frozen on dry ice, and stored at -80°C.
We used the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Cat#634894) to reverse transcribe poly(A) RNA and amplify cDNA according to the manufacturer’s instructions. We performed reverse transcription and cDNA amplification for 18 PCR cycles in 8-well strips, in sets of 12-24 strips at a time. At least 1 control strip was used per amplification set, which contained 4 wells without cells and 4 wells with 10 pg control RNA. Control RNA was either Mouse Whole Brain Total RNA (Zyagen, MR-201) or control RNA provided in the SMART-Seq v4 kit. All samples proceeded through NexteraXT DNA Library Preparation (Illumina FC-131-1096) using NexteraXT Index Kit V2 Set A (FC-131-2001). NexteraXT DNA Library prep was performed according to manufactorer’s instructions except that the volumes of all reagents including cDNA input were decreased to 0.4× or 0.5× by volume. Details are available in Documentation on the Allen Institute data portal at: http://celltypes.brain-map.org/.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description processed data file:
cells_exon_counts.csv.gz
cells_intron_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”.
Cells with fewer than 2,000 detected genes (count > 0) and less than 30% of reads mapped to the transcriptome were removed. Doublets were removed by first classifying cells into broad classes of excitatory, inhibitory, and non-neuronal based on known markers. For each class, we selected highly specific genes that are only present in this class against all other classes, and computed the eigengene (the first principle component based on the given gene set). Eigengenes for all classes were normalized in the range of 0 and 1, and we removed any cells that scored > 0.2 in more than one class.
We mapped all non-genome-mapped reads to sequences in Extended Data Table 6 (Tasic et al., 2018). To avoid ambiguous counting due to stretches of sequence identity, we designated unique regions within these sequences to count mRNAs of interest. We counted only reads for which at least one of the paired ends had an overlap with the unique regions of at least 10 bp.
Genome_build: mm10
Supplementary_files_format_and_content:
cells_*_counts.csv.gz: matrix file of exon or intron counts of non-control samples
controls_*_counts.csv.gz: matrix file of exon or intron counts of control samples
 
Submission date Jun 13, 2018
Last update date Oct 31, 2018
Contact name Allen Institute
Organization name The Allen Institute for Brain Science
Street address 615 Westlake Ave N
City Seattle
State/province WA
ZIP/Postal code 98109
Country USA
 
Platform ID GPL17021
Series (1)
GSE115746 Shared and distinct transcriptomic cell types across neocortical areas
Relations
BioSample SAMN09425052
SRA SRX4213464

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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