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Sample GSM7156247

Query DataSets for GSM7156247

Status

Public on Oct 31, 2023

Title

M1_Meadow (Multiome Gene Expression)

Sample type

SRA

Source name

primary motor cortex

Organism

Callithrix jacchus

Characteristics

tissue: primary motor cortex
species: Marmoset
donor id: Meadow
age: 6y6m
Sex: M
library type: 10x Genomics Multiome Gene Expression

Extracted molecule

total RNA

Extraction protocol

[nuclei preparation protocol] Brain tissue was pulverized using a mortar and pestle on dry ice and pre-chilled with liquid nitrogen. Pulverized brain tissue was resuspended in 1mL of chilled NIM-DP-L buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl pH 7.5, 1mM DTT, 1X Protease Inhibitor (Pierce), 1U/μL Recombinant RNase inhibitor (Promega, PAN2515), and 0.1% Triton X-100). Tissue was Dounce homogenized with a loose pestle (5-10 strokes) followed by a tight pestle (15-25 strokes) or until solution was uniform. Nuclei were filtered using a 30μm CellTrics filter (Sysmex, 04-0042-2316) into a LoBind tube (Eppendorf, 22431021) and pelleted (1000 rcf, 10 min at 4˚C) (Eppendorf, 5920 R). Pellet was resuspended in 1 mL NIM-DP buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl pH 7.5, 1mM DTT, 1x Protease Inhibitor, 1U/μL Recombinant RNase inhibitor) and pelleted (1000 rcf, 10 min at 4˚C). Pelleted nuclei were resuspended in 400uL 2μM 7-AAD (Invitrogen, A1310) in Sort Buffer (1mM EDTA, 1U/μL Recombinant RNase inhibitor, 1X Protease Inhibitor, 1% fatty acid-free BSA in PBS).
120,000 nuclei were sorted (Sony, SH800S) into a LoBind tube containing Collection Buffer (5U/uL Recombinant RNase inhibitor, 1X Protease Inhibitor, 5% fatty acid-free BSA in PBS). 5X Permeabilization Buffer (50mM Tris-HCl pH 7.4, 50mM NaCl, 15mM MgCl2, 0.05% Tween-20, 0.05% IGEPAL, 0.005% Digitonin, 5% fatty acid-free BSA in PBS, 5mM DTT, 1U/μL Recombinant RNase inhibitor, 5X Protease Inhibitor) was added for a final concentration of 1X. Nuclei were incubated on ice for 1 minute, then centrifuged (500 rcf, 5min at 4C). Supernatant was discarded and 650uL of Wash Buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1%.Tween-20, 1% fatty acid-free BSA in PBS, 1mM DTT, 1U/μL Recombinant RNase inhibitor, 1X Protease Inhibitor) was added without disturbing the pellet followed by centrifuging (500 rcf, 5 min at 4˚C). Supernatant was removed, and the pellet was resuspended in 7uL of 1X Nuclei Buffer (Nuclei Buffer (10x Genomics), 1mM DTT, 1 U/μL Recombinant RNase inhibitor). 1 μL of nuclei was diluted in 1X Nuclei Buffer, stained with Trypan Blue (Invitrogen, T10282) and counted. 16-20k nuclei were used for tagmentation reaction and controller loading and libraries were generated following manufacturer’s recommended protocol (https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression).
10x multiome ATAC-seq and RNA-seq libraries were paired-end sequenced on NextSeq 500 and NovaSeq 6000 to a depth of ~50,000 reads per cell for each modality.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

M1_Meadow_barcodes.tsv.gz
M1_Meadow_features.tsv.gz
M1_Meadow_matrix.mtx.gz

Data processing

Raw sequencing was processed using cellranger-arc (10x Genomics), generating snRNA-seq UMI count matrices for intronic and exonic reads mapping in the sense direction of a gene.
Cells were filtered for low quality nuclei by requiring ≥1000 ATAC fragments and ≥500 genes detected per nuclei.
Genes were annotated with hg38 Gencode v33 for human, mm10 Gencode vM22 for mouse, ensembl release 104 (and Refseq GCF_003339765.1 for 10x multiome) for macaque, and GCA_009663435.2 for marmoset. To maximize the number of orthologous protein-coding quantified in macaque 10x multiome RNA data, we supplemented any missing protein coding genes in GCF_003339765.1 gtf with annotations present in Ensembl release 104.
Assembly: hg38 GRCh38
Assembly: mm10 GRCm38
Assembly: Mmul_10 (rheMac10)
Assembly: cj1700_1.1 (calJac4)
Supplementary files format and content: "barcode.tsv.gz" contains cell barcode information. "features.tsv.gz" consists of rows containing features (genes and peaks) and columns containing cell-associated barcodes. "matrix.mtx.gz" contains sparse feature-barcode matrix. Files provided in tar archives.

Submission date

Apr 07, 2023

Last update date

Oct 31, 2023

Contact name

Bing Ren

E-mail(s)

biren@health.ucsd.edu

Organization name

UCSD

Street address

9500 Gilman Drive