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| Status |
Public on Jun 23, 2021 |
| Title |
133155-hg19: Human snRNA no FANS rep 1 |
| Sample type |
SRA |
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| Source name |
Skeletal muscle
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Skeletal muscle
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| Extracted molecule |
total RNA |
| Extraction protocol |
Three separate pieces of tissue were cut from a single human skeletal muscle sample (weighing 60mg, 50mg and 50mg; quadriceps femoris muscle group). Nuclei were isolated using a modified version of the ENCODE protocol (https://github.com/porchard/muscle-protocols/blob/master/Supplemental_Protocol_S1.pdf) (The ENCODE Project Consortium 2012, 2012), customized from Step 5 onwards to accommodate FANS (Fluorescence assisted nuclei sorting). In step 5, the nuclei were resuspended in 700 µL of Sort buffer (1% BSA, 1mM EDTA in PBS) and filtered through a 30 µm filter. Three different nuclei isolations were performed and the nuclei suspended in sort buffer were mixed, pooled together and divided into two groups, one with FANS and one without FANS. FANS nuclei were sorted according to the previously published FANS protocol using DRAQ7 (Preissl et al. 2018). DRAQ7 (0.3mM from Cell Signaling Technology) was added to the FANS nuclei suspension, at 100 fold dilution to get a final concentration of 3 μM. Nuclei were gently mixed and incubated for 10 minutes on ice. Nuclei were analyzed in the presence of DRAQ7 and sorted for high DRAQ7 positive signal using Beckman Coulter’s Astrios MoFlo. We followed the gating strategy outlined in the FANS protocol (Preissl et al, 2018). The sorted nuclei were collected in a recovery buffer (5% BSA in PBS). The nuclei with and without FANS were spun at 1000g for 15 min at 4°C. The nuclei were resuspended in 100 µL of 1X diluted nuclei buffer and counted in the Countess II FL Automated Cell Counter. The appropriate amount of nuclei were split for snRNA-seq and spun down at 500g for 10 min at 4°C and resuspended in RNA nuclei buffer (1%BSA+PBS in 0.2U RNAse inhibitor).
The nuclei were submitted to the Universiy of Michigan Advanced Genomics core for processing on the 10x Genomics Chromium platform (v. 3.1 chemistry for snRNA-seq). Nuclei were loaded at 15.4K nuclei/well.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
***Raw data are unavailable due to patient privacy concerns***
nuclear RNA
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| Data processing |
snRNA-seq data was processed using starSOLO (STAR v. 2.7.3a; options --soloUMIfiltering MultiGeneUMI --soloCBmatchWLtype 1MM_multi_pseudocounts --soloCellFilter None). For human-only libraries we used the GENCODE v. 19 annotation (with hg19). For human-rat mix libraries we processed the data twice, once for human and once for rat, and used the NCBI Rattus norvegicus Annotation Release 106 with rat genome rn6.
We filtered the mapped BAM file using samtools view -h -b -q 255 -F 4 -F 256 -F 2048
We anonymized the BAM files using anonymizeBAM (--strict; v. 0.4.5)
We generated single-end BED files using bedtools bamtobed
RNA-seq coverage files were generated from the anonymized BAM file using pybedtools (Dale et al., 2011) (a python wrapper around BEDTools; BedTool().genome_coverage() function, with arguments bg=True, split=True, and strand and scale set appropriately to produce both reverse and forward strand bedGraph files scaled to 1 million reads), followed by conversion of bedGraph files to bigWig files using bedGraphToBigWig (v. 4)
Genome_build: hg19 (human) and rn6 (when rat nuclei were included in the library)
Supplementary_files_format_and_content: RNA-seq read mapping locations, anonymized (BED files)
Supplementary_files_format_and_content: RNA-seq signal tracks for forward and reverse strands (bigWig files)
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| Submission date |
Jun 23, 2021 |
| Last update date |
Jun 23, 2021 |
| Contact name |
Stephen C.J. Parker |
| Organization name |
University of Michigan
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| Street address |
100 Washtenaw Avenue
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE178734 |
Human and rat skeletal muscle single-nuclei multi-omic integrative analyses nominate causal cell types, regulatory elements, and SNPs for complex traits [snRNA-seq] |
| GSE178735 |
Human and rat skeletal muscle single-nuclei multi-omic integrative analyses nominate causal cell types, regulatory elements, and SNPs for complex traits |
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| Relations |
| BioSample |
SAMN19836038 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5396679_133155-hg19.bed.gz |
6.5 Gb |
(ftp)(http) |
BED |
| GSM5396679_133155-hg19.fwd.bw |
517.3 Mb |
(ftp)(http) |
BW |
| GSM5396679_133155-hg19.rev.bw |
507.6 Mb |
(ftp)(http) |
BW |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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