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Sample GSM1752996

Query DataSets for GSM1752996

Status

Public on Aug 01, 2017

Title

Alzheimer's Brain Sample42

Sample type

SRA

Source name

NCI - MED

Organism

Homo sapiens

Characteristics

diagnosis: Alzheimer's disease
tissue: brain

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

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

Submission date

Jul 01, 2015

Last update date

May 15, 2019

Contact name

Shahar Barbash

E-mail(s)

barbashshahar@gmail.com

Organization name

The Rockefeller University

Lab

Tom Sakmar