 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 12, 2021 |
| Title |
C2C12_short_9kC |
| Sample type |
SRA |
| |
|
| Source name |
C2C12
|
| Organism |
Mus musculus |
| Characteristics |
celltype: myoblast, myotube
cell line: C2C12
library prep protocol: scRNA-seq protocol; Single Cell Whole Transcriptome Kit (Parse Biosciences).
|
| Treatment protocol |
Differentiation of C2C12, ENCODE (https://www.encodeproject.org/documents/b42974fd-1490-4d51-bdcf-7db97a3e8fe0/@@download/attachment/C2C12_Wold_protocol.pdf)
To differentiate myoblasts to myotubes, cells at 90-100% confluency were rinsed with 1X PBS and myoblast growth media was replaced with 10 mL differentiation media: high-glucose DMEM with L-glutamine and without sodium pyruvate, supplemented with 2% donor horse serum, 100 units/mL penicillin, 100 ug/mL streptomycin, and freshly-added 1uM insulin. Differentiation media was replaced every 24 hours for 3 days. ENCODE protocol: https://www.encodeproject.org/documents/b42974fd-1490-4d51-bdcf-7db97a3e8fe0/@@download/attachment/C2C12_Wold_protocol.pdf
|
| Growth protocol |
C2C12 cells (ATCC, CRL-1772) were cultured on 10 cm plates in 10 mL myoblast growth media: high-glucose DMEM with L-glutamine and without sodium pyruvate, supplemented with 20% fetal bovine serum, 100 units/mL penicillin, and 100 ug/mL streptomycin. Cells were maintained at 20-50% confluency at 37C with 5% CO2 and passaged at 1:3 or 1:4 every 2 to 3 days.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were rinsed with 1X PBS and incubated with 2 mL TrypLE-Express for 5 minutes at 37C, which was then neutralized with 8 mL myoblast growth media. RNA was reverse transcribed in situ and barcoded cDNA was extracted following Parse Biosciences’ Single Cell Whole Transcriptome Kit (also called Split-seq).
PacBio long-Split-seq: SMRTbell Template Prep Kit (PacBio, 100-938-900) following manufacturer's protocol with 500 ng of amplified, barcoded cDNA. Illumina Split-seq: Parse Biosciences’ Single Cell Whole Transcriptome Kit (also called Split-seq).
scRNA-seq
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
C2C12 myoblast cells, myoblast nuclei, and myotube nuclei were collected for Split-seq and dispersed into 48 wells of a 96 well plate containing unique barcodes per well (12 wells of myoblast cells, 12 wells of myoblast nuclei, and 24 wells of myotube nuclei). Library preparation was performed following manufacturer’s instructions for the Single Cell Whole Transcriptome Kit, where the cells are mixed for the next 2 sets of barcoding reactions followed by sublibrary generation. A mix of 9,000 cells was set aside for this sublibrary which was processed into cDNA followed by shallow sequencing with Illumina short reads.
|
| Data processing |
PacBio long-Split-seq: : Reads were processed into circular consensus reads using the CCS software from the SMRT analysis software suite (--skip-polish --min-length=10 --min-passes=3 --min-rq=0.9 --min-snr=2.5) (https://github.com/PacificBiosciences/ccs)
PacBio long-Split-seq: Split-seq adapters were identified with Lima (v2.0.0) (--ccs --min-score 0 --min-end-score 0 --min-signal-increase 0 --min-score-lead 0) (https://github.com/pacificbiosciences/barcoding/)
PacBio long-Split-seq: Reads were then run through IsoSeq3's Refine (v3.4.0) to yield full-length non-chimeric reads, without the polyA tail requirement, as half of the Split-seq primers used are random hexamer (https://github.com/PacificBiosciences/IsoSeq)
PacBio long-Split-seq: Reads were demultiplexed using a custom version of the Split-seq pipeline modified to work with long reads. (https://github.com/fairliereese/pacbio-splitpipe)
PacBio long-Split-seq: Reads were mapped with minimap2 (v2.17-r94) (-ax splice:hq -uf --MD)
PacBio long-Split-seq: Long-read sequencing artifacts were corrected with TranscriptClean (--canonOnly --primaryOnly)
PacBio long-Split-seq: Reads were run through TALON to annotate each read to its gene and transcript of origin with GENCODE vM21 (cb_tag branch on GitHub, https://github.com/mortazavilab/TALON/tree/cb_tag) (--cb)
PacBio long-Split-seq: Genes were quantified by using TALON's abundance matrix tool and aggregating counts for each gene
PacBio long-Split-seq: Transcripts were filtered for those that were reproducible across cells using TALON's filtering options.
PacBio long-Split-seq: Filtered transcripts were used to make the transcript-level abundance matrix using TALON's abundance matrix module
PacBio long-Split-seq: Filtered transcripts were used to create the transcriptome GTF of transcripts that were present across the entire dataset.
PacBio long-Split-seq: Scanpy was used to cluster, normalize, and generate AnnData h5ad gene and transcript-level expression matrices.
Illumina Split-seq: Reads were demultiplexed into 7 sublibraries using bcl2fastq and the provided adapter sequences.
Illumina Split-seq: Parse Biosciences' analysis software was run on the C2C12_short_1k sublibrary: split-pipe --mode all --genome_dir genomes/mm10/ --fq1 expdata/C2C12_short_1k_R1.fastq.gz --fq2 expdata/C2C12_short_1k_R2.fastq.gz --output_dir analysis/C2C12_short_1k --sample MB_cells A1-A12 --sample MB_nuclei B1-B12 --sample MT_nuclei C1-D12
Illumina Split-seq: Parse Biosciences' analysis software was run on the C2C12_short_9kA sublibrary: split-pipe --mode all --genome_dir genomes/mm10/ --fq1 expdata/C2C12_short_9kA_R1.fastq.gz --fq2 expdata/C2C12_short_9kA_R2.fastq.gz --output_dir analysis/C2C12_short_9kA --sample MB_cells A1-A12 --sample MB_nuclei B1-B12 --sample MT_nuclei C1-D12
Illumina Split-seq: Parse Biosciences' analysis software was run on the C2C12_short_9kB sublibrary: split-pipe --mode all --genome_dir genomes/mm10/ --fq1 expdata/C2C12_short_9kB_R1.fastq.gz --fq2 expdata/C2C12_short_9kB_R2.fastq.gz --output_dir analysis/C2C12_short_9kB --sample MB_cells A1-A12 --sample MB_nuclei B1-B12 --sample MT_nuclei C1-D12
Illumina Split-seq: Parse Biosciences' analysis software was run on the C2C12_short_9kC sublibrary: split-pipe --mode all --genome_dir genomes/mm10/ --fq1 expdata/C2C12_short_9kC_R1.fastq.gz --fq2 expdata/C2C12_short_9kC_R2.fastq.gz --output_dir analysis/C2C12_short_9kC --sample MB_cells A1-A12 --sample MB_nuclei B1-B12 --sample MT_nuclei C1-D12
Illumina Split-seq: Parse Biosciences' analysis software was run on the C2C12_short_9kD sublibrary: split-pipe --mode all --genome_dir genomes/mm10/ --fq1 expdata/C2C12_short_9kD_R1.fastq.gz --fq2 expdata/C2C12_short_9kD_R2.fastq.gz --output_dir analysis/C2C12_short_9kD --sample MB_cells A1-A12 --sample MB_nuclei B1-B12 --sample MT_nuclei C1-D12
Illumina Split-seq: Parse Biosciences' analysis software was run on the C2C12_short_9kE sublibrary: split-pipe --mode all --genome_dir genomes/mm10/ --fq1 expdata/C2C12_short_9kE_R1.fastq.gz --fq2 expdata/C2C12_short_9kE_R2.fastq.gz --output_dir analysis/C2C12_short_9kE --sample MB_cells A1-A12 --sample MB_nuclei B1-B12 --sample MT_nuclei C1-D12
Illumina Split-seq: Parse Biosciences' analysis software was run on the C2C12_short_9kF sublibrary: split-pipe --mode all --genome_dir genomes/mm10/ --fq1 expdata/C2C12_short_9kF_R1.fastq.gz --fq2 expdata/C2C12_short_9kF_R2.fastq.gz --output_dir analysis/C2C12_short_9kF --sample MB_cells A1-A12 --sample MB_nuclei B1-B12 --sample MT_nuclei C1-D12
Genome_build: mm10
Supplementary_files_format_and_content: Long-split-seq Scanpy h5ad file with normalized gene or transcript-level expression values
Supplementary_files_format_and_content: GTF of long-Split-seq transcript model structure
Supplementary_files_format_and_content: For Illumina Split-seq, Parse Biosciences analysis software (split-pipe) outputs are sparse cell-by-gene expression matrices in counts with corresponding cell metadata and a list of expressed genes (columns of expression matrix).
|
| |
|
| Submission date |
Mar 11, 2021 |
| Last update date |
Mar 13, 2021 |
| Contact name |
Elisabeth Rebboah |
| E-mail(s) |
erebboah@uci.edu
|
| Organization name |
University of California, Irvine
|
| Department |
Developmental and Cell Biology
|
| Lab |
Mortazavi Lab
|
| Street address |
2300E Biological Sciences 3
|
| City |
Irvine |
| State/province |
California |
| ZIP/Postal code |
92617 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE168776 |
Mapping and modeling the genomic basis of differential RNA isoform expression at single-cell resolution with LR-Split-seq |
|
| Relations |
| BioSample |
SAMN18274943 |
| SRA |
SRX10327378 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5169187_C2C12_short_9kC.genes.mtx.gz |
35.9 Mb |
(ftp)(http) |
MTX |
| GSM5169187_C2C12_short_9kC_cell_metadata.csv.gz |
146.9 Kb |
(ftp)(http) |
CSV |
| GSM5169187_C2C12_short_9kC_genes.csv.gz |
222.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
