Specimens

Animals and sample preparation: Sprague Dawley rats (Taconic Farms, Germantown, NY) were used and were maintained on a 14:10 light:dark (L:D) cycle. Animal use and care protocols were approved by the NIH Institutional Animal Care and Use Committee and followed the guidelines of the National Research Council's Guide for Care and Use of Laboratory Animals (Vol. 8) and the Animal Research: Reporting In vivo Experiments (ARRIVE) guidelines. Timed pregnant animals were used; on postnatal day 5 male and female pups were divided into the four surgical groups and subjected to the respective surgical interventions.

Surgery: The neonatal decentralization (DCN) and superior cervical ganglionectomy (SCGX) procedures were based on modifications to previously published procedures for adult rats (1). Five-day-old rat pups were anesthetized (Isoflurane), placed supine under a surgical microscope and the ventral neck region was disinfected with povidone-iodine. The salivary glands were exposed through a 1-cm vertical incision and retracted laterally to expose the underlying muscles. The carotid bifurcations were identified through the carotid triangle and the SCG were dissected bluntly. Further steps consisted of: a) SCGX group: SCG were removed after sectioning successively all its nervous branches with micro-dissecting spring scissors; b) DCN group: the cervical sympathetic trunks were dissected bluntly and severed 0.5 mm before entering the SCG and at 4 to 5mm caudally, removing 4 to 5 mm of the cervical sympathetic chain; c) Sham group: no further steps. Following completion of the surgical procedures on both sides of the neck the wound was cleansed and then closed with VetBond adhesive. Survival rate was 100%.

Animals were then maintained in a 14:10 L:D cycle until day 40. The four surgical groups were: no surgical intervention (Control); neonatal sham surgery (Sham); neonatal bilateral DCN; SCGX (1). Each surgical group had 18 to 20 animals, which were then divided into two sub groups. At 40 days of age animals were euthanized by CO2 asphyxiation and decapitated. Animals sacrificed at ZT19 were decapitated in dim red light. Samples were pooled into three biological replicates for each of the eight biological conditions (24 pooled samples, total). Glands were immediately placed in microtubes on solid CO2 (three glands per tube) and stored at -80C until use.

RNA Preparation

RNA extraction: Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA), followed by clean-up using an RNeasy Micro Kit with on-column DNase treatment as per the manufacture's protocol (Qiagen, Valencia, CA). The amount and quality of RNA were determined using a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Each pool yielded 2.1 to 3.7 micrograms of total RNA (RIN values > 9).

RNA-Seq library preparation and sequencing

Library preparation and sequencing: Stranded RNA-Seq libraries were constructed from 0.7-1ug total RNA using the TruSeq Stranded Total RNA Sample Prep Kits (Illumina cat. no. RS-122-2301) according to manufacturer's instructions. The library insert sizes were approximately 175bp. Unique barcode adapters were applied to each library. Equal volumes of individual libraries were pooled and run on a MiSeq (Illumina, San Diego, CA). The libraries were then repooled based on the MiSeq demultiplexing results. This combined library was sequenced on six lanes of Illumina HiSeq 2000 sequencer using version 3 chemistry. This yielded an average of 48.6 million read-pairs for each sample (ranging from 42.9 million to 56.4 million).

Bioinformatics methods

See bioinformatics methods for details.

References

1. Savastano LE, Castro AE, Fitt MR, Rath MF, Romeo HE, Munoz EM. A standardized surgical technique for rat superior cervical ganglionectomy J Neurosci Methods. 2010;192(1):22-33. Epub 2010/07/20.

2. Hartley SW, Coon SL, Savastano LE, Mullikin JC; NISC Comparative Sequencing Program, Fu C, Klein DC. Neurotranscriptomics: The Effects of Neonatal Stimulus Deprivation on the Rat Pineal Transcriptome PLoS One. 2015 Sep 14;10(9):e0137548. doi: 10.1371/journal.pone.0137548. eCollection 2015