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Sample GSM999581

Query DataSets for GSM999581

Status

Public on Oct 04, 2012

Title

Parkinson's Disease PD 7

Sample type

SRA

Source name

Brain cortex

Organism

Homo sapiens

Characteristics

tissue: Human brain cortex (BA9)
disease state: Parkinson's Disease PD
gender: F
age at death (y): 87

Treatment protocol

None

Growth protocol

None

Extracted molecule

polyA RNA

Extraction protocol

RNA was extracted from frozen brain tissue using miRNeasy kits (Qiagen) and quantified using a Nanodrop (Thermo). First-strand cDNA was synthesized from 1 μg of RNA per biological sample using SuperScript III (Invitrogen) following manufacturer’s instructions and using the pdT-FS oligonucleotide to prime the reverse transcription. Barcoded first-strand samples from different samples were then pooled and treated with RNase H (Invitrogen) at 37°C for 20 minutes followed by 15 minutes at 75°C to degrade RNA template. First-stand cDNA was then purified using QIAquick PCR Purification kit (Qiagen) in a total volume of 30uL. Second-strand cDNA was synthesized from 25uL of first-strand cDNA template by adding 10 μl 10x buffer 2 (NEB), 5 μl 10 mM dNTPs, 20 U Klenow Fragment (3’ → 5’exo-; NEB), 10 μl of 100 μM tagged 2nd strand primer (R-SS oligonucleotide: 5’-TCCGATCTGANNNNNNN-3’ with N=A,C,T,G mix) and 46 μl water. The reaction mix was incubated at 37°C for 30 minutes, followed by 10 minutes at 75°C then cooled down at 4°C. Double-stranded cDNA was purified using PureLink PCR micro columns (Invitrogen) in a 30uL volume. Illumina-compatible libraries were then generated by PCR from 25uL of double-stranded cDNA template using Accuprime Pfx polymerase (Invitrogen) following manufacturer’s instruction with NNSR forward (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCT-3’) and NNSR reverse (5’-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGA-3’) primers. Thermo-cycling conditions were 2 min at 94 °C followed by 2 cycles of 94 °C for 10 s, 40 °C for 2 min, 68 °C for 1 min; 8 cycles of 94 °C for 10 s, 60 °C for 30 s, 68 °C for 1 min; 15 cycles of 94 °C for 15 s, 60 °C for 30 s, 68 °C for 1 min with an additional 10 s added at each cycle; and 68 °C for 5 min before cooling to 4 °C. Amplified libraries were purified using PureLink PCR micro columns (Invitrogen) and directly used to generate clusters for sequencing-by-synthesis using the Illumina HiSeq 2000 platform. 100bp single-end reads were obtained by sequencing to generate more than 300 million reads for the 34 samples.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Sample 23

Data processing

Illumina reads were converted to FASTQ Sanger format using FASTQ Groomer (Galaxy)
The first 27 bp at their 5’ends of the reads were trimmed using FASTQ Trimmer to remove the polyA and adapters sequences
The reads were mapped to human hg19 genome using Burrows-Wheeler Alignment tools with Galaxy’s default settings allowing 4% of missing alignments.
Genome_build: hg19
Supplementary_files_format_and_content: Processed files contain the quantification for each sample of the 5 3'UTR isoforms detected for alpha-synuclein

Submission date

Sep 08, 2012

Last update date

May 15, 2019

Contact name

Herve RHINN

E-mail(s)

hr2239@columbia.edu

Phone

2123051150

Fax

2123051150