GIS_Prenatal_Midbrain_7
Homo sapiens
Protocols: Total RNAs were isolated from either hMLOs, 2D-DA neurons, or undifferentiated hESCs using TRIzol Reagent (Invitrogen). DNA contaminants were digested with DNASE I (Ambion). The ventral half of the midbrain was dissected from second trimester prenatal brainstem under visual inspection, using a scalpel and hand-held dental drill. Cases were donated with informed consent of the mother, and were selected on the basis of the absence of any congenital anatomical abnormality on macroscopic brain inspection, and the absence of any major genetic defect noted on prenatal testing. The IRB approval is from the Western IRB, a private and fully accredited IRB that is used by many independent institutions in the USA: address 1019 39th Avenue SE Suite 120, Puyallup, WA 98374-2115. Study number: 1126332. WIRB protocol number: 20111080. Approval expires: 7/18/2016 RNA-seq libraries were constructed using Illumina TruSeq Stranded Total RNA Ribo-Zero sample Prep Kit following the manufacturer's protocol. The ribosome RNAs were removed using Ribo-zero beads from ~ 800 ng DNAse treated total RNA. Following purification, the total RNA without Ribosome RNA was fragmented into small pieces using divalent cations under elevated temperature (94 degree) for 2 minutes. Under this condition, the range of the fragments length is from 130-290 bp with a median length of 185 bp. Reverse transcriptase and random primers were used to copy the cleaved RNA fragments into first strand cDNA. The second strand cDNA was synthesized using DNA Polymerase I and RNaseH, dUTP in place of dTTP. These cDNA fragments then went through an end repair process using T4 DNA polymerase, T4 PNK and Klenow DNA polymerase, and the addition of a single 'A' base using Klenow exo (3' to 5' exo minus), then ligation of the Illumina PE adapters using T4 DNA Ligase. An index (96 unique dual-index pairs) was inserted into Illumina adapters so that multiple samples can be sequenced in one lane of 8-lane flow cell if necessary. These products were then purified and enriched with 15 cycles of PCR to create the final cDNA library for high through put DNA sequencing using the H 100 base-pair paired-end reads and the Illumina HiSeq 2000, targeting approximately 100M reads per sample. The concentration of RNA libraries was measured by Qubit (Invitrogen, CA). The quality of RNA-seq library was measured by LabChipGX (Caliper, MA) using HT DNA 1K/12K/HiSens Labchip.
ENA first public
2016-08-24
ENA last update
2016-06-27
External Id
SAMEA4051987
INSDC center alias
GIS
INSDC center name
Genome Institute of Singapore
INSDC first public
2016-08-24T17:08:01Z
INSDC last update
2016-06-27T18:04:29Z
INSDC status
public
Submitter Id
E-MTAB-4868:GIS_Prenatal_Midbrain_7
broker name
ArrayExpress
common name
human
developmental stage
prenatal
organism part
midbrain
sample name
E-MTAB-4868:GIS_Prenatal_Midbrain_7
sex
female