RNA-Seq of mus musculus: hippocampus at Postnatal 8 days; NRSF-cKO mice
Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen)/RNeasy Mini Kit(Qiagen)/other kits. Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer(Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrosegel. 1 g total RNA with RIN value above 6.5 was used for following library preparation. Next generationsequencing library preparations were constructed according to the manufacturers protocol. The poly(A)mRNA isolation was performed using Poly(A) mRNA Magnetic Isolation Module or rRNA removal Kit.The mRNA fragmentation and priming was performed using First Strand Synthesis Reaction Buffer andRandom Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and thesecond-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified doublestrandedcDNA by beads was then treated with End Prep Enzyme Mix to repair both ends and add a dAtailingin one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of AdaptorligatedDNA was then performed using beads, and fragments of ~420 bp (with the approximate insert sizeof 300 bp) were recovered. Each sample was then amplified by PCR for 13 cycles using P5 and P7primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCRand P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned upusing beads, validated using an Qsep100 (BiopticTaiwan China), and quantified by Qubit3.0Fluorometer (Invitrogen, Carlsbad, CA, USA).