title: NextSeq 550 paired end sequencing; Bulk RNA-seq of paired human brown and white adipocytes
design: Bulk RNA-seq of paired human brown and white adipocytes
library_construction_protocol: Human adipose tissue was obtained from the central compartment of the neck, superior to the clavicle and deep to the lateral thyroid lobe either adjacent to the longus colli muscle or to the oesophagus (brown adipose tissue; BAT) and more superficially from the subcutaneous neck tissue (white adipose tissue; WAT). The stromal vascular fraction was isolated and cultured as described (Ramage, Akyol et al. 2016 doi: 10.1016/j.cmet.2016.06.011). Following differentiation, cells were cultured in serum-stripped medium for 48 hours prior to RNA extraction and subsequent bulk RNA-seq.   Sample 91 – age (years): 41; sex: female; height (m): 1.52; weight (Kg): 63.5; BMI (kg/m2): 27.48 Sample 92 – age (years): 59; sex: female; height (m): 1.57; weight (Kg): 60.4; BMI (kg/m2): 24.50 Sample 93 – age (years): 52; sex: female; height (m): 1.70; weight (Kg): 114; BMI (kg/m2): 39.45 Sample 95 – age (years): 50; sex: female; height (m): 1.72; weight (Kg): 66; BMI (kg/m2): 22.31 Total RNA samples were assessed on the Agilent Bioanalyser (Agilent Technologies, #G2939AA) with the RNA 6000 Nano Kit (#5067-1511) for quality and integrity of total RNA. Samples were DNase-treated using the TURBO DNA-free™ kit (Thermo Fisher, #AM1907) and then quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc, #Q32866) and the Qubit RNA BR assay kit (#Q10210), and assessed for residual DNA contamination using the Qubit dsDNA HS assay Kit (catalogue # Q32851). Libraries were prepared from each TURBO™ DNase treated total-RNA sample using the TruSeq Stranded Total RNA with Ribo-Zero kit (#RS-122-2201) according to the provided protocol. 100ng of total-RNA was processed to deplete rRNA before being purified, fragmented and primed with random hexamers. Primed RNA fragments were reverse transcribed into first strand cDNA using reverse transcriptase and random primers. RNA templates were removed and a replacement strand synthesised incorporating dUTP in place of dTTP to generate ds cDNA. AMPure XP beads (Beckman Coulter, #A63881) were then used to separate the ds cDNA from the second strand reaction mix, providing blunt-ended cDNA. A single 'A' nucleotide was added to the 3' ends of the blunt fragments to prevent them from ligating to another during the subsequent adapter ligation reaction, and a corresponding single 'T' nucleotide on the 3' end of the adapter provided a complementary overhang for ligating the adapter to the fragment. Multiple indexing adapters were then ligated to the ends of the ds cDNA to prepare them for hybridisation onto a flow cell, before 15 cycles of PCR were used to selectively enrich those DNA fragments that had adapter molecules on both ends and amplify the amount of DNA in the library suitable for sequencing.  Libraries were quantified using the Qubit dsDNA HS kit and by PCR using the Kapa Universal Illumina Library Quantification kit complete kit (#KK4824) and assessed for quality using the Agilent Bioanalyser with the DNA HS Kit.