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Status |
Public on Aug 01, 2017 |
Title |
Alzheimer's Brain Sample49 |
Sample type |
SRA |
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Source name |
DEM - LOW
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Organism |
Homo sapiens |
Characteristics |
diagnosis: Alzheimer's disease tissue: brain
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted with the SPLIT RNA extraction kit (Lexogen GmbH, Vienna, Austria) yielding the large RNA fraction with a lower cut-off size of 150 nt. Evaluation on a Bioanalyzer RNA Nano chip (Agilent Technologies) showed medium to high RNA quality (RIN of 6.2 – 8.3). 4 µg RNA was incubated with Terminator 5´-phosphate-dependent exonuclease (Epicentre, Madison, WI, USA) to remove degraded RNA while leaving the capped, full-length mRNA intact Per sample, 20 ng of the purified full-length total RNA were used for performing the 3’ QuantSeq Reverse protocol (Lexogen GmbH, Vienna, Austria) resulting in NGS libraries which originate from the 3’ end of polyadenylated RNA. Library generation was started by oligo(dT) priming with primers already containing the Illumina-compatible linker sequence for Read 1. After first strand synthesis the RNA was removed before the second strand synthesis was initiated by random primers which contained the corresponding Illumina-compatible linker sequence. 3’ QuantSeq generated just one fragment per transcript at the very 3’ end. The libraries were PCR amplified and barcoded in 18 cycles. Equal amounts of the 72 libraries were combined to one lane mixture.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Reference genome alignment: sequence reads in fastq format were aligned to the reference genome GRCh38 from Ensembl, repeat-masked version, complete genome, using the bowtie2 aligner version 2.2.3 and the –end-to-end and –sensitive options, to insure global alignments of the query sequences. Alignment conversion: sequence alignment conversion from textual .sam to binary .bam files was performed with samtools version 0.1.19 Sequence trimming and primer removal was performed with the bbduk.sh utility from the BBMap project (http://sourceforge.net/projects/bbmap/). Reference file were a long polyA tail and the sequences of the Illumina truseq primer. Options: ktrim=r (trim right end), useshortkmers=t (look for shorter kmers at read tips), mink=5 (minimum length of short kmers), trimq = 10 (regions with average quality below this value will be trimmed), qtrim=t (trim reads to remove bases below both ends). Genome_build: GRCh38
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Submission date |
Jul 01, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Shahar Barbash |
E-mail(s) |
barbashshahar@gmail.com
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Organization name |
The Rockefeller University
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Lab |
Tom Sakmar
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Street address |
1230 York Ave
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City |
New York |
State/province |
NY |
ZIP/Postal code |
10065 |
Country |
USA |
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Platform ID |
GPL16791 |
Series (1) |
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Relations |
BioSample |
SAMN03835362 |
SRA |
SRX1078681 |
Supplementary data files not provided |
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Raw data are available in SRA |
Processed data are available on Series record |
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