title: GSM5215984: Ctl Tet2+/- RNA rep1; Mus musculus; RNA-Seq
library_construction_protocol: RNA-seq: Nasal mucosa samples were homogenized in TRIzol (Invitrogen, cat# 15596026) and processed according to the manufacturer's instructions. RNA-seq: RNA was then reverse transcribed using a SuperScript III First-Strand Synthesis System (Thermo Fisher, cat# 18080051). cDNA was quantified by qPCR using TaqMan Universal PCR Master Mix (Thermo Fisher, cat# 4304437) and SYBR Green PCR Master Mix (Thermo Fisher, cat# 4309155). Each reaction was run in triplicate and analyzed following the ΔΔCT method using glyceralde-hyde-3-phosphate dehydrogenase (Gapdh) as normalization control. ChIP-seq: Genomic DNA was isolated from nasal tissues in 550μl of digestion buffer (100 mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl), then treated with Proteinase K (Thermo) at 55 °C overnight. The second day, 550μl of phenol: chloroform: isoamyl alcohol (25:24:1 saturated with 10 mM Tris, pH 8.0, 1 mM EDTA) (P-3803, Sigma) was added to samples, mixed completely, and centrifuged for 10 min at 12,000 rpm. The aqueous layer was transferred into a new Eppendorf tube and precipitated with 600μl of isopropanol. The pellet was washed with 75% ethanol, air-dried, and resuspended with Nuclease-Free Water (Ambion). DNA samples were treated extensively with an RNase cocktail (Ambion) to remove contaminating RNA. ChIP-seq: DNA libraries were generated following the Illumina protocol for “Preparing Samples for ChIP Sequencing of DNA” (Part# 111257047 Rev. A) using 25–50 ng of input genomic DNA or 5hmC-captured DNA to initiate the protocol. All sequencing libraries were run on Illumina Hi-seq 2000 machines. FASTQ sequence files from biological replicates were concatenated and aligned to the M. musculus reference genome (GRCm38/mm10) using Bowtie2 v2.3.5.139, keeping unique non-duplicate genomic matches with no more than 2 mismatches within the first 25 bp.