RNA-seq from surface library Round 1, mouse 2, Brain
RNA isolation from snap-frozen tissues was performed within one week of the in vivo study. Frozen tissues were ground in liquid nitrogen with a mortar and pestle over dry ice to remain frozen. A subset of the ground tissue was weighed, dissolved in TRIzol, sonicated with a Sonic Dismembrator 60 (Fisher Scientific), and stored at -80C until RNA extraction. Samples were stored in TRIzol (Invitrogen 15596026) at -80C. RNA was purified from samples using RNeasy Kits (Qiagen 74104 or 74106) according to the manufacturers instructions. RNA and DNA concentrations were measured with NanoDrop or Qubit (Invitrogen Q32852, Q32854). Reverse transcription (PrimeScript 1st strand cDNA Synthesis Kit, Takara 6110A), quantitative PCR (KAPA HiFi HotStart Ready Mix, Roche; SYBR Green, Invitrogen S7563), and sequencing on the Illumina MiSeq (Illumina MiSeq Reagent Kit v3, MS-102-3003; 5% PhiX Control v3, FC-110-3001) were performed as previously described and/or according to manufacturers instructions unless otherwise noted (Butterfield & Lajoie, 2017, Nature) The skpp-132-R reverse primer was used for reverse transcription instead of the primers and 6mers provided by the Takara PrimeScript 1st strand cDNA Synthesis Kit (5-CATACTGTTGGTTGCTAGGC-3). The skpp-132-F forward primer was used to amplify synNC genomes with qPCR (5-TAGGATTACTGCTCGGTGAC-3). The skpp-offset-R reverse primer was used to amplify synNC genomes with qPCR (5-GTTGCTAGGCTCAGTGATGG-3).