organism: Homo sapiens
description: 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.
attributes.tag: ENA first public
attributes.value: 2016-08-24
attributes.tag: ENA last update
attributes.value: 2016-06-27
attributes.tag: External Id
attributes.value: SAMEA4051982
attributes.tag: INSDC center alias
attributes.value: GIS
attributes.tag: INSDC center name
attributes.value: Genome Institute of Singapore
attributes.tag: INSDC first public
attributes.value: 2016-08-24T17:08:01Z
attributes.tag: INSDC last update
attributes.value: 2016-06-27T18:04:29Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4868:GIS_Prenatal_Midbrain_2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: prenatal
attributes.tag: organism part
attributes.value: midbrain
attributes.tag: sample name
attributes.value: E-MTAB-4868:GIS_Prenatal_Midbrain_2
attributes.tag: sex
attributes.value: male