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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 12, 2020 |
| Title |
Col_r1_InDrop |
| Sample type |
SRA |
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| Source name |
Colon
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| Organism |
Mus musculus |
| Characteristics |
age (weeks): 6
strain: Phox2b-CFP
Sex: Male
fixation: Fixed
platform: inDrop
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Nuclei were isolated using the NucleiEZ lysis buffer with modifications, as described. FACS was performed to isolate neuronal nuclei and glia, which were gated for 7AAD+ and high intensity of CFP from the Phox2b-CFP reporter and collected in Eppendorf LoBind protein plates coated with 1% BSA in 1X PBS.
[10x] For generation of 10X libraries, nuclei were encapsulated using version 3 Chromium Single Cell 3’ library reagents. Libraries were sequenced using a Nova-seq 6000 or Nextseq 500 and a paired-end 50 bp sequencing flow cell at a total depth of 3.6 Billion reads across all 10X runs
[inDrop] inDrop utilizes CEL-Seq in preparation for sequencing and is summarized as follows: 1) Reverse transcription (RT) (supplemented with 30µM DTT), 2) ExoI nuclease digestion (no HinFI), 3) SPRI purification (SPRIP), 4) Second strand synthesis, 5) T7 in vitro transcription linear amplification, 6) SPRIP, 7) short RNA fragmentation, 8) SPRIP, 9) Primer annealing, 10) RT, 11) SPRIP and 12) library enrichment PCR (doubled reaction size), 13) SPRIP. Number of nuclei encapsulated was calculated by observing the single nuclei suspension loading rate multiplied by bead loading efficiency during the duration of encapsulation. The largest sample contained an estimated 5000 nuclei, while the smallest contained an estimated 100 nuclei. Libraries were sequenced on a Nextseq 500 (Illumina) with a 150bp paired-end sequencing kit in a customized sequencing run.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Run3316
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| Data processing |
[10x] The sequencing output FASTQs were processed with CellRanger 3.0.2 and aligned to the reference transcriptome (intron+exon)
[inDrop] For inDrop, reads were filtered, sorted by their designated barcode, and aligned to the reference transcriptome (intron + exon) using DropEST pipeline (STAR). Mapped reads were quantified into UMI-filtered counts per gene.
Genome_build: mm10
Supplementary_files_format_and_content: matrix (inDrop): contains a gene-barcode-count matrix
Supplementary_files_format_and_content: matrix (10x): contains a raw count matrix
Supplementary_files_format_and_content: features (10x): contains a list of detected genes with Ensembel IDs and gene symbols corresponding to the associated 10x matrix file
Supplementary_files_format_and_content: barcodes (10x): contains an ordered list of barcodes corresponding to the 10x matrix file
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| Submission date |
Jun 24, 2020 |
| Last update date |
Oct 12, 2020 |
| Contact name |
E Michelle Southard-Smith |
| E-mail(s) |
michelle.southard-smith@vanderbilt.edu
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| Phone |
615-293-4236
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| Organization name |
Vanderbilt University Medical Center
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| Department |
Medicine, Genetic Medicine
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| Street address |
2215 Garland Ave,
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| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE153192 |
Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ [Mouse sn-RNA-Seq] |
| GSE153202 |
Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ |
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| Relations |
| BioSample |
SAMN15360448 |
| SRA |
SRX8610692 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4635430_3316-MS-4-GTGAAA_S4_cm_x.csv.gz |
1.6 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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