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| Status |
Public on Oct 03, 2019 |
| Title |
Left ventricle tissue of end-stage DCM patients (20LB01727) |
| Sample type |
SRA |
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| Source name |
Left ventricle tissue of end-stage DCM patients
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Left ventricle tissue
sequencing: Ribo-seq
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| Extracted molecule |
total RNA |
| Extraction protocol |
Briefly, snap-frozen cell pellets or 50-100 mg of tissue, previously powdered under liquid nitrogen, were lysed in 1ml cold lysis buffer (formulation as in TruSeq Ribosome Profile, Illumina) supplemented with 0.1mg/ml cycloheximide (CHX) to stabilize ribosomal subunits and prevent post-lysis translocation. Homogenized and cleared lysates were then footprinted with Truseq Nuclease (Illumina) according to manufacturer’s instructions. Ribosomes were purified using Illustra Sephacryl S400 columns (GE Healthcare) and the protected RNA fragments were extracted with standard phenol:chloroform:isoamylalcohol technique.
Following ribosomal RNA removal (Mammalian RiboZero Magnetic Gold, Illumina), sequencing libraries were prepared out of the footprinted RNA. Ribo-seq libraries were pooled to perform multiplex sequencing on Illumina Hiseq machines.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Ribosome protected fragment
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| Data processing |
Library strategy: Ribo-seq
Raw sequencing data were demultiplexed with bcl2fastq V2.19.0.316 and the adaptors were trimmed using Trimmomatic19 V0.36, retaining reads longer than 20 nt post-clipping. RNA-seq reads were further clipped with FASTX Toolkit V0.0.14 to 29nt, to allow comparison directly with Ribo-seq reads. Reads were aligned using bowtie20 to known rRNA, tRNA and mt-rRNA sequences (RNACentral21, release 5.0), aligned reads were filtered out to obtain only RPFs (Ribosome protected fragments). Alignment to the human genome (hg38) was done using STAR22. Gene expression was quantified on the CDS (coding sequence region) using uniquely mapped reads (Ensembl database release GRCh38 v86 combined with additional transcripts from RefSeq GRCh38, latest version downloaded January 2018) with feature counts23.
Genome_build: hg38
Supplementary_files_format_and_content: count matrix for protein coding genes across 30 samples
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| Submission date |
May 13, 2019 |
| Last update date |
Oct 03, 2019 |
| Contact name |
Sonia Chothani |
| E-mail(s) |
[email protected]
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| Organization name |
Duke-NUS school of medicine
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| Lab |
Prof. Stuart Cook
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| Street address |
8 College Road
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| City |
Singapore |
| ZIP/Postal code |
169857 |
| Country |
Singapore |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE131111 |
Translational control of cardiac fibrosis (II) |
| GSE131112 |
Translational control of cardiac fibrosis |
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| Relations |
| BioSample |
SAMN11635164 |
| SRA |
SRX5825537 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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