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Status |
Public on Nov 24, 2020 |
Title |
2011-325 RNA84 [control] |
Sample type |
SRA |
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Source name |
corneal endothelium
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Organism |
Homo sapiens |
Characteristics |
group: Control tissue: corneal endothelium batch: 2
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Extracted molecule |
total RNA |
Extraction protocol |
RNA libraries were prepared using TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA). Briefly, poly(A) mRNA was purified from total RNA using oligo(dT) magnetic beads, fragmented at 95 °C for 8 min, eluted from the beads, and primed for first-strand cDNA synthesis using SuperScript III reverse transcriptase and random primers (Invitrogen). Second- strand cDNA synthesis was performed using DNA polymerase I and RNase H, and double-stranded cDNA was purified using a single AMPure XP bead cleanup step (Agencourt, Danvers, MA). The cDNA ends were repaired and phosphorylated using Klenow fragment, T4 polymerase, and T4 polynucleotide kinase, followed by a single AMPure XP bead cleanup step. The blunt-ended cDNAs were modified to include a single 3 -adenylate residue using Klenow exo (3 to 5 exo ), and paired-end DNA adaptors (Illumina) with a single T base overhang at the 3 -end were ligated to the A-tailed cDNA population. Unique indexes, included in the standard TruSeq kits (12- Set A and 12-Set B), were incorporated at the adaptor ligation step for multiplex sample loading on the flow cells. The resulting constructs were purified by two consecutive AMPure XP bead cleanup steps and enriched by 12 cycles of PCR using primers included in TruSeq RNA Sample Prep Kit v2. Libraries were loaded onto paired-end flow cells at concentrations of 8–10 pM to generate cluster densities of 700,000/mm2 using cBot and cBot Paired-end Cluster Kit v3 (Illumina) following the manufacturer’s standard protocol. The flow cells were sequenced as 51 2 paired-end reads on an Illumina HiSeq 4000 system using TruSeq SBS Sequencing Kit v3 and SCS v1.4.8 data collection software.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Data processing |
Base calling Illumina CASAVA v1.8.2 QC was performed on the FASTQ files using RSeQC software (http://rseqc.sourceforge.net/) The sequencing reads were processed by MAP-RSeq v.1.2 workflow [1], the Mayo Bioinformatics Core pipeline. The alignment in MAPRseq was performed with TopHat 2.0.6 and the gene counts was obtained using HTSeq-count v0.5.3p9 Genome_build: hg19 Supplementary_files_format_and_content: tab delimited file includes raw gene counts
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Submission date |
Nov 23, 2020 |
Last update date |
Nov 24, 2020 |
Contact name |
Xiaojia Tang |
E-mail(s) |
Tang.Xiaojia@mayo.edu
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Organization name |
Mayo Clinic
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Street address |
200 1st ST SW
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City |
Rochester |
State/province |
MN |
ZIP/Postal code |
55905 |
Country |
USA |
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Platform ID |
GPL20301 |
Series (1) |
GSE112201 |
RNA Misplicing in Fuchs Endothelial Corneal Dystrophy II |
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Relations |
BioSample |
SAMN16879009 |
SRA |
SRX9565494 |
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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