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Sample GSM5523065

Query DataSets for GSM5523065

Status

Public on Sep 08, 2021

Title

SM-GE67F_S061_E1-50

Sample type

SRA

Source name

Macaque LGN Magnocellular

Organism

Macaca nemestrina

Characteristics

donor: Q18.27.001
Sex: M
age: 2.8 yrs
dissection_roi: MC
facs gating: NeuN-positive
sequencing batch: R8S4-180228
cell class: Glut
cell cluster: M

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 14/10-h day/night cycle at no more than five adult animals per cage. Animals were maintained on the C57BL/6J background. 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. Cux2-CreERT2;Ai140 mice received tamoxifen treatment at P35 ± 5 for five consecutive days. We excluded any animals with anophthalmia or microphthalmia. The brain tissues of two adult M. nemestrina (southern pig-tailed macaque) and one M. fascicularis (crab-eating macaque) were obtained through the Tissue Distribution Program of the Washington National Primate Research Center and conformed to the guidelines provided by the US National Institutes of Health. All procedures were approved by the Institutional Animal Care and Use Commit- tee of the University of Washington under protocol number 4277-01.

Extracted molecule

polyA RNA

Extraction protocol

We isolated single cells and nuclei as previously described (Tasic et al., 2016, Bakken et al., 2018) with modifications. 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. For nuclei isolation, dissected regions of interest were transferred to microcentrifuge tubes, snap frozen in a slurry of dry ice and ethanol, and stored at -80°C until the time of use. To isolate nuclei, frozen tissues were placed into a homogenization buffer that consisted of 10mM Tris pH 8.0, 250mM sucrose, 25mM KCl, 5mM MgCl2, 0.1% Triton-X 100, 0.5% RNasin Plus RNase inhibitor (Promega), 1X protease inhibitor (Promega), and 0.1mM DTT. Tissues were placed into a 1ml dounce homogenizer (Wheaton) and homogenized using 10 strokes of the loose dounce pestle followed by 10 strokes of the tight pestle to liberate nuclei. Homogenate was strained through a 30μm cell strainer (Miltenyi Biotech) and centrifuged at 900xg for 10 minutes to pellet nuclei. Nuclei were then resuspended in staining buffer containing 1X PBS supplemented with 0.8% nuclease-free BSA and 0.5% RNasin Plus RNase inhibitor.
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

Data processing

Processing of SSv4 libraries was performed as described previously (Tasic et al., 2018). Briefly, libraries were sequenced on an Illumina HiSeq2500 platform (paired-end with read lengths of 50 bp) to a target read depth of 0.5M reads per cell (range 100,275-12,329,698, median 1,003,867). The Illumina sequencing reads were aligned to GRCm38.p3 (mm10) using a RefSeq annotation gff file retrieved from NCBI on 18 January 2016
Sequence alignment was performed using STAR v2.5.3. (Dobin et al., 2013) in the two-pass mode. 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) and summarizeOverlaps function in ‘IntersectionNotEmpty’ mode for exonic and intronic regions separately. For the SSv4 dataset, we only used exonic regions for gene quantification. Cells that met any one of the following criteria were removed: < 100,000 total reads, < 1,000 detected genes (with CPM > 0), < 75% of reads aligned to genome, or CG dinucleotide odds ratio > 0.5. Doublets were removed by first classifying cells into broad classes of excitatory, inhibitory, and non-neuronal based on known markers. 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) and were used as a QC metric.
Genome_build: mm10, Mmul_10

Submission date

Aug 16, 2021

Last update date

Sep 08, 2021

Contact name

Bosiljka Tasic

E-mail(s)

bosiljkat@alleninstitute.org

Organization name

Allen Institute for Brain Science

Department

Molecular Genetics