Ribo zero goldkits

Total RNA from the hypothalamus was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, United States). RNA purity was checked with a Nano Photometer^® spectrophotometer (IMPLEN, Westlake Village, CA, United States), and its concentration was measured with Qubit^® RNA Assay kits (Thermo Fisher Scientific). RNA integrity was assessed with RNA Nano 6000 Assay, with the RNA integrity number (RIN) value of all samples being greater than seven.
Three micrograms of total RNA were taken as a starting amount to construct a lncRNA library. To remove ribosomal RNA (rRNA) from the sample, Ribo-Zero^TM GoldKits (Epicentre, United States) were used to digest the total RNA. In addition, in accordance with the manufacturer’s instructions for the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, United States), RNA-sequencing libraries were generated by paired-end sequencing. Subsequently, the pooled libraries were sequenced on Hiseq X (Illumina, San Diego, CA, United States), using a chain-specific library construction strategy to count the number and types of various transcripts (mRNAs, known lncRNAs and novel lncRNAs). All sequencing data was outsourced to Annoroad Gene Technology Co., Ltd. (Beijing, China).

Total RNA was isolated from the left ventricular tissues of mice with TRIzolTM reagent (Invitrogen, United States). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, United States) prior to library preparation. The qualified RNA samples were treated with Ribo-Zero^TM Gold Kits (Epicenter Technologies, United States) to remove ribosomal RNA (rRNA). Sequencing libraries were generated using the NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, United States) following the manufacturer’s recommendations. The library preparations were sequenced on an Illumina HiSeq 4000 platform, and 125 bp/150 bp paired-end reads were generated.

Pituitary PT tissues were used for RNA extraction with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. Examination involving the integrality and quality of the isolated RNA was performed via electrophoresis and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).
The rRNA was depleted from 3 μg of total RNA using Ribo-Zero TM Gold Kits (Epicentre, Madison, WI, USA). Sequencing libraries of the 12 samples (SP21, n = 3; LP7, n = 3; LP21, n = 3; LP42, n = 3) were generated using NEB Next Ultra Directional RNA LibraryPrep Kit for Illumina (NEB, Ipswich, MA, USA) according to the manufacturer's instructions, and index codes were used to label the sequences of each sample. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform (Illumina, San Diego, CA, USA). Raw data of the performed RNA-seq have been recorded in the SRA public database (Accession number of BioProject: PRJNA680667).