title: Illumina HiSeq 4000 paired end sequencing; Comparison of the myometrial transcriptome from singleton and twin pregnancies by high throughput RNA-Seq
design: Comparison of the myometrial transcriptome from singleton and twin pregnancies by high throughput RNA-Seq
library_construction_protocol: Samples of myometrium were collected by knife biopsy from the lower uterine segment surgical incision from women undergoing elective caesarean section under spinal anesethesia. Samples were placed in cold hanks balanced salt solution, transported to the laboratory where myometrial tissue was carefully dissected under light microscope.  Myometrium (approx 200mg) was placed into RNA later for 24 hours at 4oC before being frozen at -80C until RNA extraction. Myometrial tissue ( 90-100mg) was homogenised using a homogeniser and an RNA purification kit involving Trizol and choloroform (Trizol plus RNA purification kit - invitrogen). Briefly tissues were homogensied in 1mL trizol, followed by the addition of 0.2mL chloroform. Phase separation was performed at 12000g at  4oC for 15 minutes.  RNA phase was added to by equal volumes of 70% ethanol ( final concnetration 35%) before binding, washing and elution on column as per kit instructions.  Eluted RNA was stored at -80C. To remove traces of genomic DNA, samples underwent DNAse treatment using the Ambion Turbo DNA-free kit. This  was followed by an RNAClean XP bead purification step  to ensure that all traces of the DNase inactivation reagent was removed.  Samples were quantified by Qubit, and purity and integrity examined by nanodrop and later by abn agilent bioanalyser to determine RIN above >7.0. Total RNA samples were treated with Turbo DNase (Life Technologies). Samples were incubated with enzyme for 30 minutes at 37°C before the reaction was terminated with the supplied slurry. RNA was recovered using Agencourt RNA clean beads (1.8 vol beads : 1 vol sample) and checked for quantity with Qubit assay and quality using an Agilent Bioanalyser chip. 1 μg of DNA-free  total RNA for each sample was poly-A selected using NEBNext® Poly(A) mRNA Magnetic Isolation Module. Then sample library was prepaired using the NEBNext® UltraTM Directional RNA Library Prep kit following manufactuer's instructions. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyser. These final libraries were pooled in equimolar amounts using the Qubit and Bioanalyzer data. The quantity and quality of each pool was assessed by Bioanalyzer and subsequently by qPCR using the Illumina Library Quantification Kit from Kapa (KK4854) on a Roche Light Cycler LC480II according to manufacturer's instructions.