title: GSM8705610: 5xFAD-ASSIST4; Mus; RNA-Seq
library_construction_protocol: Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure.  The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA).  The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel. Poly (A) RNA is purified from 1μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) using two rounds of purification.   Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.  e6150, USA) under 94℃ 5-7min.   Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat.   1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.  m0209, USA), RNase H (NEB, cat.  m0297, USA) and dUTP Solution (Thermo Fisher, cat.  R0133, USA）.   An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters.   Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA.   Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads.   After the heat-labile UDG enzyme (NEB, cat.  m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min;   8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec;   and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50 bp.   At last, we performed the 2×150bp paired-end sequencing (PE150) on an illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.