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Sample GSM3629671

Query DataSets for GSM3629671

Status

Public on Apr 01, 2019

Title

8RC2HUA

Sample type

SRA

Source name

muscle and endothelial cells

Organisms

Homo sapiens; Mus musculus

Characteristics

cell type: C2C12 mouse myoblasts and human microvascular endothelial cells (HMEC-1)
passages: 10 to 15
strain: C3H

Growth protocol

Mouse myoblasts (C2C12, ATCC) were expanded in a high serum DMEM with 20% FBS. Human microvascular ECs (Center for Disease Control) were expanded in 10% FBS and 1% penicillin/streptomycin in DMEM. All cell culture was performed with 1% penicillin/streptomycin at 37°C and 5% CO2. Where indicated, mouse myoblasts were lentivirally transduced to express GFP under the control of ubiquitin promoter. For in vitro studies, four conditions were assessed: 1) randomly-oriented scaffold seeded with myoblasts; 2) randomly-oriented scaffolds seeded with myoblasts + ECs; 3) aligned scaffold seeded with myoblasts only; 4) aligned scaffold seeded with myoblasts + ECs. Collagen scaffold strips (25mm x 1mm) were sterilized with 70% ethanol and rehydrated with 3 washes with 1X PBS. Mouse myoblasts (5x105) were seeded into each scaffold and cultured in a high serum DMEM with 20% FBS. After 24 hours (Day 0), the growth medium was switched to DMEM containing 2% horse serum to induce differentiation and fusion of myoblasts. After 5 days of differentiation (Day 5) during which myoblasts have fused to form myotubes, ECs were cocultured into the myoblast-seeded scaffolds (5x105 cells/scaffold). For conditions with myoblasts only, no ECs were added. For all conditions, media was switched to a maintenance medium (DMEM supplemented with 2% horse serum and 3% FBS), and constructs were culture for an additional 4 days (Day 9).

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from cell lysates from engineered muscle and endothelialized engineering muscle on randomly-oriented or aligned scaffolds (n=3) according to the manufacture’s protocol (GeneJet RNA Purification Kit, Thermo Fisher). At least 1000ng of total RNA was pelleted per sample.
Library preparation and RNA Sequencing was performed by Novogene Corporation using the NovaSeq 6000 (Illumina) sequencing platform, and 150-bp paired-end reads were generated (20-30 million reads per sample). Briefly, RNA integrity was assessed (RNA Nano 6000 Assay Kit, Bioanalyzer 2100, Aligent Technologies, CA) and 3ug RNA per sample was used to generate sequencing libraries (NEBNext Ultra RNA Library Prep Kit for Illumina) according to manufacturer’s recommendations. Library fragments were purified (AMPure XP system, Beckman Coulter, Beverly) to select cDNA fragments (150-200 bp in length). PCR was performed with Phusion High-Fidelity DNA polymerase, and purified by the AMPure XP system and assessed for quality on the Agilent Bioanalyzer 2100 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

diffgene_AC_vs_RC_gsea_rank.txt

Data processing

The raw data were checked for quality with FastQC (Version 0.11.7) and results were aggregated with MultiQC and were aligned to the mouse genome (GRCm38) using STAR (Version 2.5.3a) with ENCODE options for long RNA-seq pipeline.
The alignment results were assessed using Samtools and aggregated with MultiQC (Version 1.5) and the differential gene expression analysis of the uniquely mapped reads/raw counts was performed using the DESeq2 package (Version 1.20.0).
Each DE analysis was composed of a pairwise comparison between an experimental group and the control group. Differentially expressed genes were identified after false discovery rate (FDR = 0.05) correction.
Genome_build: mouse genome (GRCm38)
Supplementary_files_format_and_content: txt files contain DE comparison analysis as described above

Submission date

Feb 26, 2019

Last update date

Apr 01, 2019

Contact name

Ngan Huang

E-mail(s)

ngantina@stanford.edu

Phone

5108476870

Organization name

Stanford University