Purezol

Total RNA was isolated from mouse ventricles using PureZOL^TM (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. RNA from each sample was used for real time (RT) using the iScript cDNA synthesis kit (Bio-Rad). The cDNA was used for quantitative polymerase chain reaction (PCR) analysis in an amplification system (MX3000P, Stratagene, La Jolla, CA, USA) using Brilliant III Ultra-Fast SYBR^® Green QPCR Master Mix (Agilent, Santa Clara, CA, USA). Primers used for RT-PCR were as follows:

RyR2 forward 5′-CTGAGAACTGATGATGAGGTGGT-3′;

RyR2 reverse 5′-ATCCTTCTGCTGCCAAGCAC-3′;

18S forward 5′CGGACAGGATTGACAGATTG-3′;

18S reverse 5′-CAAATCGCTCCACCAACTAA-3′;

NOX4 forward 5′-TGGCCAACGAAGGGGTTAAA-3′;

NOX4 reverse 5′-ATGAGGCTGCAGTTGAGGTT-3′;

NOX2 forward 5′-CTCAGGCCAATCACTTTGCT-3′;

NOX2 reverse 5′-TTCAGGGCCACACAGGAAAA-3′.

The 2^−ΔΔCt method was used to calculate relative transcript abundances.

Cortices were lysed in 500 µl of Purezol^TM (Bio‐Rad). RNA was isopropanol precipitated and treated with DNAse I (Sigma‐Aldrich), re‐extracted with phenol‐chloroform and isopropanol precipitated over night at −20°C. RNA pellets were dissolved in 20 µl of Nuclease free water (Sigma‐Aldrich). RNA quantity was measured using NanoDrop. 1,000 ng of RNA was then retro‐transcribed using the RT² First Strand kit (Qiagen). qRT–PCR results depicted in Fig 5 and in the Appendix Fig S3 were performed using SYBR selected master mix (Applied Biosystems) as fluorescent dye. Primers used for the analyses are reported in Appendix Table S1. Excel (Microsoft) and Prism (GraphPad) were used to elaborate data.

Cells were rinsed once with PBS and lysed in Purezol^TM (300 µl for roughly 30,000 cells; Bio‐Rad). RNA was isopropanol precipitated and treated with DNAse I (Sigma‐Aldrich), re‐extracted with phenol‐chloroform and isopropanol precipitated over night at −20°C. RNA pellets were dissolved in 10 µl of TE buffer. RNA concentration and quality were checked using Bioanalyzer RNA 6000 Nano chips (Agilent). 50 ng of total RNA was reverse‐transcribed using Superscript IV Reverse Transcriptase (Thermo Fisher). 1 ng of cDNA for each sample was pre‐amplified using a 0.2X pool of 74 primers (TaqMan; Thermo Fisher; Appendix Table S1) for 18 cycles using PreAmp Master Mix (Fluidigm) to enable multiplex sequence‐specific amplification of targets. Pre‐amplified cDNAs were then diluted and assessed using a 96 × 96 qPCR Dynamic Array microfluidic chip (Fluidigm) following the manufacturer’s instructions. Baseline correction was set on Linear (Derivative) and Ct threshold method was set on Auto (global). To normalize, for each sample a “pseudogene” (D’haene et al,2012) was obtained by averaging the Ct value of each gene depicted in Appendix Table S1 except for Mecp2. Each sample was then normalized against its “pseudogene.” Excel, Prism, and R were used to elaborate data.

Using a homogenizer, the hippocampal tissues were combined with 1000 µL of RNA extraction solution (PureZOLTM: BioRad, Hercules, California, USA). Then 400 µL of chloroform was added, and a 15-second vortex was performed. They were kept for 15 minutes at room temperature before being centrifuged at 14 000 g for 20 minutes at +4°C. A new Eppendorf tube was used to contain the aqueous phase that had developed at the end of the centrifugation. Depending on the volume of the aqueous phase, 300 µL of isopropanol was added, and the container was repeatedly turned upside down. They were centrifuged for 30 minutes after being kept at room temperature for 10 minutes. At the end of the centrifugation, the supernatant portion was discarded. Before vortexing, 1 mL of 75% ethanol was added to the particle that was still at the bottom. It was then centrifuged for 5 minutes at +4°C at 7500 g. After discarding the supernatant part created after centrifugation, ethanol surrounding the pellet was removed with a pipette. The particle was dried for 10-15 minutes at room temperature, allowing the ethanol to evaporate. Twenty microliter of nuclease-free water (NFW)was added after the drying procedure, and the particle was resuspended. It was covered with ice and stored at +4°C for 10-15 minutes. RNA concentration (ng/µL) was measured in the NanoDrop at the end of this procedure.

Cells were rinsed once with PBS and lysed in Purezol^TM (300 µl for roughly 30,000 cells; Bio‐Rad). RNA was isopropanol precipitated and treated with DNAse I (Sigma‐Aldrich), re‐extracted with phenol‐chloroform and isopropanol precipitated over night at −20°C. RNA pellets were dissolved in 10 µl of TE buffer. RNA quantity was checked using NanoDrop (Thermo Fisher Scientific). 100 ng of RNA was then retro‐transcribed using the RT² First Strand kit (Qiagen). qRT–PCR results depicted in the Fig EV2 were performed using SYBR green (Life Technologies) as fluorescent dye. Primers used for the analyses are reported in Appendix Table S1. Excel (Microsoft) and Prism (GraphPad) were used to elaborate data.

Hair follicles preserved into PureZOL™ were subjected to RNA extraction with Aurum™ Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad), following the manufacturer’s instructions.
The quality and quantity of total RNA extracts were measured using NanoDropTM spectrophotometer (Thermo Fisher Scientific,). Furthermore, the microfluidic electrophoresis on the BioAnalyzer 2100 (Agilent Technologies, California, USA) revealed the integrity and suitability of the RNA samples for NGS library preparation. Libraries were prepared according to the Illumina TruSeq2 kit, using poly-A mRNA purification. A total of 10 RNA samples, 5 for anagen and 5 for telogen respectively, were sequenced on one lane of Illumina HiSeq 1500 platform generating 150 base-paired end reads, according to the TruSeq2 kit.