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| Status |
Public on Apr 01, 2024 |
| Title |
Amygdalostriatal transition area biological replicate 2 |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: brain
tissue region: amygdalostriatal transition area (ASt)
Sex: male
age: 8 weeks
strain: C57BL6/J
genotype: wild type
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
All sacrifices were performed during the dark period of the light cycle. Animals were anesthetized via combined intraperitoneal injection of 150 mg/kg ketamine and 15 mg/kg xylazine. Once unconscious, mice animals were transcardially perfused with ice-cold, carbogen-bubbled (95% O2, 5% CO2), nuclease-free, 0.22 µm sterile-filtered artificial cerebrospinal fluid (ACSF) with a composition of 93 mM N-methyl-D-glucamine, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 13.2 mM trehalose, 12 mM N-acetyl-cysteine, 0.5 mM CaCl2, 10 mM MgSO4, and 93 mM HCl, at pH 7.3-7.4.(Tasic et al., 2018; Ting et al., 2014) Following transcardial perfusion, brains were immediately extracted and submerged into ice-cold carbogen-bubbled ACSF, with less than 5 minutes between the beginning of perfusion and final submersion after extraction. Brains were serially sectioned in ice-cold, carbogen-bubbled ACSF on a VT1000S vibratome (Leica) with polytetrafluoroethane-coated razor blades (Ted Pella) at 0.15 mm/sec and 100 Hz, dividing the whole cerebrum into 400 µm coronal slices. Target regions were microdissected from these slices under a stereomicroscope using a sterile blunt-end needle (22 gauge for CeA, ASt, and tail of striatum, 16 gauge for dorsal striatum). All regions were targeted using Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates as reference (Paxinos and Franklin, 2019). Extracted tissue samples were recovered in ice-cold, nuclease-free, 0.22 µm sterile-filtered cryoprotective nuclear storage buffer, composed of 0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 10 mM Trizma hydrochloride buffer (pH 8.0), 1 mM dithiothreitol, 0.02 U/µl SUPERase•In RNAse Inhibitor (Invitrogen), and 1X cOmplete Protease Inhibitor Cocktail with EDTA (Roche). Tissue was then snap frozen using a metal CoolRack M90 (Biocision) pre-chilled to -80˚C and stored at -80˚C until nuclear isolation. Cryopreserved tissue pieces were slow-thawed by incubation at 4˚C for 1 hour prior to isolation. Tissue pieces were then pooled and resuspended in nuclear isolation medium composed of 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Trizma hydrochloride buffer (pH 7.4), 1 mM dithiothreitol, 0.04 U/µl RNasin Plus RNAse Inhibitor (Promega), 1X cOmplete Protease Inhibitor Cocktail with EDTA (Roche), and 0.1% Triton-X. The pooled tissue pieces in nuclear isolation medium were transferred to a 2 mL Dounce tissue grinder. Tissue was homogenized by 5 strokes from the loose pestle and 15 followed by the tight pestle, and the resulting homogenate was filtered through a 40 µm Flowmi cell strainer (Bel-Art) into a 1.5 ml Lo-Bind tube (Eppendorf). The homogenate was then centrifuged with a swinging bucket rotor at 4˚C and 1000 x g for 8 minutes. Nuclei were then washed with nuclear flow buffer composed of DPBS with 1% bovine serum albumin, 1 mM dithiothreitol, and 0.04 U/µl RNasin Plus RNAse Inhibitor (Promega) and centrifuged at 4˚C and 500 x g for 5 minutes, which was subsequently repeated. Nuclei were finally resuspended in nuclear flow buffer containing 3 µm DRAQ7 (Cell Signaling Technology) and again filtered through a 40 µm Flowmi cell strainer into a 5 ml round-bottom polystyrene tube. Each isolation took under 45 minutes to perform, from homogenization to final suspension.
Library construction was performed acording to manufacturers' instructions (10X Single Cell 3' v3.1 Single Index). GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip. RNA from nuclei in each droplet was reverse-transcribed to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with DynaBeads, PCR-amplified and appropriately-sized fragments were selected with SPRIselect reagents for subsequent library construction. During subsequent steps, Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
ASt-2
10X Single Cell 3' v3.1 Single Index
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| Data processing |
Fastq demultiplexing with CellRanger (v5.0.0)
Read alignment and single cell data parsing with CellRanger (v5.0.0)
Reads aligned to CellRanger 2020-A reference genome (mm10) and annotation (GENCODE vM23/Ensembl 98)
Empty droplets were called using the emptyDrops model from DropletUtils (v1.10.2), where droplets with less than 100 counts were used to profile ambient RNA and over 1000 were assumed non-empty
Minimal quality filtering for each barcode was performed by setting a floor of 1000 features per barcode for downstream inclusion to ensure the dataset is entirely composed of high-quality nuclei. Next, to remove highly likely multiplet barcodes, barcodes were filtered out if their count depth was more than 5 median absolute deviations above the median count depth. Barcodes were then removed if their proportion of ribosomal or mitochondrial reads was more than 5 interquartile ranges above the 75th percentile.
Heterotypic doublets were identified by creating simulated artificial doublets in scDblFinder (v1.4.1),
Assembly: mm10
Supplementary files format and content: Barcodes: Tab-separated values files containing cell barcodes and matrix files
Supplementary files format and content: Features: Tab separated values files containing the gene identities in the matrix
Supplementary files format and content: Matrix: raw barcode by feature matrix files giving counts for each combination
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| Submission date |
Aug 17, 2022 |
| Last update date |
Apr 01, 2024 |
| Contact name |
Kay Tye |
| E-mail(s) |
tye@salk.edu
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| Organization name |
Salk Institute
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| Department |
Systems Neurobiology Laboratory
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| Lab |
Tye Lab
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| Street address |
10010 N Torrey Pines Rd
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE211437 |
Amygdalostriatal transition zone neurons encode sustained valence to direct conditioned behaviors |
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| Relations |
| BioSample |
SAMN30410612 |
| SRA |
SRX17157667 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6470103_ASt_2_barcodes.tsv.gz |
18.5 Mb |
(ftp)(http) |
TSV |
| GSM6470103_ASt_2_features.tsv.gz |
254.1 Kb |
(ftp)(http) |
TSV |
| GSM6470103_ASt_2_matrix.mtx.gz |
206.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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