|
| Status |
Public on Aug 08, 2014 |
| Title |
PNFF-hMPN1 |
| Sample type |
SRA |
| |
|
| Source name |
human PN patient cells, compensated with hMPN1
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: poikiloderma with neutropenia (PN) patient
cell type: foreskin fibroblasts from PN patient (PNFF)
genotype/variation: PNFF-hMPN1; compensated with hMPN1
|
| Growth protocol |
Logarithmically growing yeast cells in liquid YES media at 30°C, shaking at 150 rpm; PNFF-derived cells were maintained in DMEM supplemented with 20% fetal bovine serum, non-essential amino acids, penicillin/streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hot phenol RNA dextraction protocol (Schmitt ME, Brown TA & Trumpower BL (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 18: 3091–3092); Trizol reagent was used to isolate total RNA from human cells.
10 µg of total RNA was ligated to Thaliana_lig_2 adaptor oligonucleotides (rArGratcggaagagcgtcgtgtagggaaagag-ddT where r denotes ribonucleotides) using Thaliana tRNA ligase. RNA was purified and treated with 20 U of rSAP (Affymetrix) in rSAP buffer (25 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 mM ATP (NEB)) at 37°C for 1 hour in order to remove 2’ phosphates remained on ligated RNA. RNA was fragmented with RNA fragmentation reagent (Ambion). The fragmented RNA was then reverse transcribed using primers complementary to the ligated RACE adaptor. the obtained cDNA was used to construct sequencing library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
GQY36
|
| Data processing |
Basecalls performed using CASAVA version 1.8.2
Reads were aligned to the human or yeast genome using RUM version 2.0.4
A reference collection consisting of RNA features and (for human samples) features from the RepeatMasker database (http://www.repeatmasker.org) was created
Each aligned read was if possible assigned to one of these features with HTSeq-count, using the "union" method to handle read alignments overlapping multiple features
Reads aligning to multiple positions in the genome were defined to contribute "1/N read" to each position
Genome_build: ASM294v1.17 (yeast), hg19 (human)
Supplementary_files_format_and_content: txt files containing the estimated expression level of each quantified feature
|
| |
|
| Submission date |
Aug 07, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Claus M Azzalin |
| E-mail(s) |
claus.azzalin@bc.biol.ethz.ch
|
| Organization name |
ETH Zürich
|
| Department |
Institute of Biochemistry
|
| Street address |
Otto-Stern-Weg 3
|
| City |
Zürich |
| ZIP/Postal code |
8093 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE60197 |
Global 3' RACE in yeast and humans |
| GSE60198 |
The Mpn1 exonuclease defines a novel spliceosomal snRNA decay pathway dependent on Rrp6 and Lsm2-8 complex |
|
| Relations |
| BioSample |
SAMN02954389 |
| SRA |
SRX671726 |
