title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq of ventricle and fat tissues of male and female cardiac myocyte-specific estrogen receptor KO mice
design: RNA-seq of ventricle and fat tissues of male and female cardiac myocyte-specific estrogen receptor KO mice
library_construction_protocol: tissues were collected at dissection, placed into 1.5ml tubes and immediately flash frozen in liquid nitrogen. Tissue samples were then stored at -80 degrees. RNA was extracted from ventricles and subcutaneous white adipose tissue (WAT) from each group using RNA-easy MiniPrep kit according to manufacturer's instructions (Qiagen). RNA quality for sequencing was determined using the high-sensitivity Agilent RNA ScreenTape Assay for 4200 Tape Station System (Agilent Technologies). Only samples with an RNA Integrity Number (RIN) score >7 were selected for subsequent analysis. DNA libraries were generated as per the manufacturer's protocol using the NEBNext Poly(A) mRNA Magnetic Isolation Module kit (New England Biolabs). 1μg total RNA per sample was inputted and adaptor ligation and PCR enrichment was performed using the NEBNext Multiplex Oligos for Illumina kit as per manufacturer's instructions. Libraries were individually bar coded using n6-barcodes and library quantity and quality were assessed as per manufacturer's instructions using the Agilent D1000 ScreenTape Assay for 4200 TapeStation System.