Agilent 2100 bionalyzer

Upon dissection, samples for qPCR were immediately embedded in RNAlater for preservation (Qiagen, Valencia, CA). Total RNA was extracted by homogenization in TRIzol (Invitrogen, Paisley, UK) following the manufacturer’s protocol. Extracted RNA was treated with RNase-free Recombinant DNaseI (Roche Diagnostics, Mannheim, DE) and RNA concentration was assessed by spectrophotometry and its quality checked using an Agilent 2100 bionalyzer (Agilent Technologies, Santa Clara, US). RNA (1.2 μg) was reverse transcribed by random primers using Affinity Script Multiple Temperature cDNA Synthesis Kit (Agilent Technologies) following the manufacturer’s protocol and then diluted 1:2 with nuclease-free water.

Bilateral L4 DRG from SNL and sham groups were harvested six days after surgery, immediately treated in RNAlater (Ambion, Austin, TX) and subjected to total RNA extraction. To obtain enough RNA for sequencing, eight L4 DRGs from four mice were pooled together and each group required three biological replicates (n = 12 mice/group). For quantitative real-time PCR assay, four L4 DRGs from the ipsilateral sides of four SNL or sham mice were pooled together and the experiment was repeated three times (n = 12 mice/group). Briefly, total RNA was extracted using the miRNeasy kit with on-column digestion of genomic DNA (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. RNA concentration was measured using the NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE) and Qubit Fluorometric Quantitation (Invitrogen, Carlsbad, CA). Ratios of A260/280 nm were between 1.97 and 2.08. RNA integrity was assessed using RNA Nano chips in an Agilent 2100 Bionalyzer (Agilent technologies, Santa Clara, CA). RNA integrity numbers (RIN) were between 7.5 and 8.4.

Spinal nerve transection (SNT) of the L5 spinal nerve was performed on male Wistar rats (n = 12) as described in [33 (link)]. L5 dorsal root ganglia (DRG) were harvested 7 days after surgery by fresh dissection, immediately frozen in liquid nitrogen and stored at -80°C. L5 DRG tissue from naive animals (n = 12) was used as control. Tissue from 4 animals was pooled to create three independent biological replicates per group (SNT or naive) and total RNA was extracted using the miRNEasy kit (QIAGEN, Redwood City, CA, USA) according to manufacturer’s instructions. RNA concentration was measured using the NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). RNA integrity was assessed using RNA Nano chips in an Agilent 2100 Bionalyzer (Agilent technologies, Santa Clara, CA, USA); RNA integrity numbers (RIN) were between 8.5 and 9.3. Each RNA sample was separated into two technical replicates; one was further processed for microarray analysis and the other for RNA-seq library preparation. Each RNA-seq library was further subdivided into 3 technical replicates, which were sequenced to three distinct read depths as described below.