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Sample GSM5909124

Query DataSets for GSM5909124

Status

Public on Jan 22, 2023

Title

p-dC_MG_in_porcine_with_VNS_3

Sample type

SRA

Source name

Myenteric ganglia from descending colon

Organism

Sus scrofa

Characteristics

strain: Yucatan minipigs
tissue: colon
age: ~7 months old
cell type: Myenteric ganglia
treatment: Electrical stimulation of the celiac branch of the abdominal vagus nerve

Treatment protocol

A detailed experimental protocol for VNS in porcine is available at https://www.protocols.io/view/tache‐mulugeta‐ot2od024899‐colon‐tissue‐electrical‐3rmgm.

Extracted molecule

total RNA

Extraction protocol

For bulk RNA-seq, a range of 25-40 ganglia/subject were harvested from ISG and MG respectively of p-pC and p-dC and from MG of h-aC and h-dC using LMD-6000 Laser Micro-dissection System. Total RNA was extracted using QIAgen RNAeasy Micro Kit. SMART-Seq® Stranded Kit was used with around 190 pg of total RNA for the construction of sequencing libraries. For preparation of cell suspensions, full thickness colon tissue samples were collected from each p-pC and p-dC of 4 naïve porcine. The tissue samples were washed in ice-cold, carbogen (95% O2 and 5% CO2)-bubbled PBS. The muscularis externa containing myenteric ganglia were peeled off from the underlying tissue using forceps, followed by incubation in enteric neuron media, containing Neurobasal A media with B-27, 2 mM L-glutamine, 1% fetal bovine serum (FBS), 10 ng/ml Glial Derived Neurotrophic Factor and 1× Antibiotic/Antimycotic, in the presence of 45 µM Actinomycin D (ActD) for 15 min on ice. The samples were then cut into small pieces < 1 mm and transferred to the enteric neuron media containing 1mg/ml collagenase B and dispase II and 45 µM ActD for 1 hour at 37°C. After the addition of 1 mg/ml deoxyribonuclease I and 10% FBS, the tissue pieces were dissociated using fire polished Pasteur pipettes. Following manual trituration, the cells were filtered through 70 µm Nitex mesh filter and pelleted at 375 g for 10 min, and resuspended in the ice-cold, carbogen-bubbled rinse medium containing F12 media with 10% FBS and 1× antibiotic/antimycotic. Importantly, the solutions used in all steps were equilibrated in the carbogen gas. Estimation of viable cell number was done by Trypan Blue dye exclusion method. After cell staining with DAPI, the viable cells were collected via fluorescence-activated cell sorting (FACS). ~10,000 single cells per cell suspension were loaded on the 10x Genomics Chromium platform accompanying Chromium™ Single Cell 3’ Library Construction and sequenced on an Illumina NextSeq 500 Sequencer.
RNA libraries were prepared for sequencing using standard protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Description

Read_counts_in_male_porcine_without_and_with_vagal_nerve_stimulation.csv

Data processing

For bulk RNA-seq data processing, the libraries derived from total RNA of ganglia in porcine were sequenced on an Illumina HiSeq 3000 sequencer as 50 base pair single-end reads. Sus_scrofa genome files (.fa) and annotation file (.gtf) were downloaded at https://uswest.ensembl.org/Sus_scrofa/Info/Index, which were used to create the sus_scrofa genome index using STAR v2.7.1a. Human reads were aligned to Ensembl release 97 human with STAR. The fastq files were then aligned against the genome assembly using STAR, followed by assessment for the total number of aligned reads and total number of uniquely aligned reads to evaluate sequencing performance, with parameters --runMode alignReads --outSAMtype BAM SortedByCoordinate Unsorted --outFilterMultimapNmax 1 --quantMode GeneCounts --twopassMode Basic --sjdbOverhang 50.
For single-cell RNA-seq data processing, Cell Ranger 3.1.0 count function was used to align and quantify the reads against sus_scrofa genome assembly with parameters --localcores=32 --expect-cells=10000 --localmem=200, created using the Cell Ranger 3.1.0 mkref function based on the downloaded Sus_scrofa genome files (.fa) and annotation file (.gtf).
Genome_build: hg38, susScr11
Supplementary_files_format_and_content: Bulk RNA-seq processed data files contain matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: Single-cell RNA-seq processed data files contain only detected cell-associated barcodes in MEX format. Each element of the matrix is the number of UMIs associated with a feature (row) and a barcode (column).

Submission date

Feb 21, 2022

Last update date

Jan 22, 2023

Contact name

Tao Li

Organization name

University of Southern California

Department

Department of Medicine

Lab

Shaker's Lab