GSM5396114: UPN_162 PWS UPD -ASD Neurons; Homo sapiens; RNA-Seq
Total RNA was extracted from ~3 week cultured Neurons using the Zymo Direct-zol RNA purification kit. Total RNA was checked for quantity and quality on an Agilent Bioanalyzer 6000 pico chip and determined to have an RNA Integrity Number (RIN) of >9.0. For eukaryotic organisms, mature mRNAs, processed and equipped with a poly(A) tail at the 3'end, are captured via oligo(dT) beads. For prokaryotic samples, rRNAs are removed by specialized kits, maintaining mRNA molecules. According to different demands, the mRNAs can be constructed into regular library and strand specific library.    Construction of regular RNA library (non-stranded, non-directional):  After enrichment, mRNAs are fragmented randomly in fragmentation buffer and the first-strand cDNA is synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). The second strand is subsequently generated by dNTPs, DNA polymerase I and RNase H. Double-stranded cDNA molecules are purified by AMPure XP beads and overhanging ends are repaired to blunt ends by exonuclease/polymerase. After 5' phosphorylation and 3' adenylation, the cDNAs are ligated with P5/P7 sequencing adapters to prepare for hybridization(1). In order to select the insert fragment of preferentially 150~200 bp in length, the modified libraries are purified with AMPure XP system (Beckman Coulter, Beverly, USA). The final library is ready after PCR amplification and products purification by AMPure XP beads.