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Sample GSM7156219 Query DataSets for GSM7156219
Status Public on Oct 31, 2023
Title M1_donor3 (Multiome Gene Expression)
Sample type SRA
Source name primary motor cortex
Organism Homo sapiens
Characteristics tissue: primary motor cortex
species: Human
donor id: H19.30.004
age: 58 y.o.
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 (
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_donor3_barcodes.tsv.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
Organization name UCSD
Street address 9500 Gilman Drive
City La Jolla
State/province CA
ZIP/Postal code 92093
Country USA
Platform ID GPL24676
Series (1)
GSE229169 Comparative single cell epigenomic analysis of gene regulatory programs in the rodent and primate motor cortex
SRA SRX19907341
BioSample SAMN34115802

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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