Cortex Untreated Rep2
The quantity and quality of the RNAs were evaluated using Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Cat.# Q32855) and Agilent RNA 6000 Nano Chips (Agilent Technologies, Cat. # 5067-1511),respectively. Sequencing libraries were prepared following TruSeq Stranded mRNA Sample Preparation Guide (Part # 15031058 Rev. E) using the TruSeq Stranded mRNA Library Prep kit (Illumina Inc. Cat. # 20020594) and TruSeqRNASingleIndexes(Illumina Inc. Cat. # 20020492 and 20020493) or TruSeq RNA CD Index Plate (96 Indexes, 96 Samples) (Illumina Inc. Cat. # 20019792). Starting from 500ng of total RNA, mRNA was purified, fragmented and primed for cDNA synthesis. cDNA first strand was synthesized with SuperScript-II Reverse Transcriptase (Thermo Fisher Scientific, Cat. # 18064-014) for 10 min at 25C, 15 min at 42C, 15 min at 70C and pause at 4C. cDNA second strand was synthesized with Illumina reagents at 16C for 1 hour. Then, A-tailing and adaptor ligation were performed. Finally, enrichment of libraries was achieved by PCR (30 sec at 98C; 15 cycles of 10 sec at 98C, 30 sec at 60C, 30 sec at 72C; 5 min at72C and pause at 4C).