GSM7071038
Endoscopic biopsies from the distal oesophagus were placed in RNA later solution (Sigma, Cat. Number R0901-100ml) and kept at -80ºC until RNA extraction. A total of 9-10 biopsy samples were selected for sequencing from each GORD phenotype. RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Cat. Number 74016). A maximum of 30 mg of tissue was used for each RNA extraction to ensure sufficient RNA yield for sequencing. Nanodrop and bioanalyzer were used to perform quality control (QC) checks of RNA supplied to the Genome Centre. RNA samples with RNA integrity less than 7 were excluded from library preparation. cDNA libraries were generated using the NEBNext Ultra II Directional RNA Library Preparation Kit with the mRNA isolation module using the manufacturer's instructions (Illumina). Briefly, RNA samples were first diluted to 20ng/μl using RNAse-free water. First strand synthesis reaction buffer was prepared and left on ice until reaction. NEBNext oligo d(T) beads were washed with RNA binding buffer, placed on a plate and subsequently resuspended in RNA binding buffer on a magnetic rack. The plate was then sealed and placed on a thermal cycler with the lid heated to 75°C, at 65°C for 5min to denature RNA and facilitate binding of the poly-A mRNA to the beads. Following centrifugation, resuspension, and washing of the beads, the plate was placed on the magnetic rack again and supernatant was discarded. Next, 50μl of Tris buffer was added per well, plate sealed and incubated at 80°C for 2min with the lid heated to 90°C. The plate was removed when the temperature reached 25°C, centrifuged, and 50μl RNA binding buffer added per well to allow mRNA to re-bind beads. The supernatant was removed using the magnetic rack, and mRNA eluted from the beads by adding 11.5μl of the first strand synthesis buffer and placing in the thermal cycler with the lid heated to 105°C for 10min at 94°C. Following completion of incubation, the plate was removed from the thermal cycler, centrifuged, and purified mRNA collected by transferring 10μl of supernatant to a clean nuclease-free PCR plate. The plate was then placed on ice and first strand cDNA synthesis was immediately started.  The first strand enzyme mastermix (composed of NEBNext strand specificity reagent and NEBNext first strand synthesis enzyme mix) was added to the fragmented and primed mRNA, gently mixed, and the plate sealed and placed in the thermal cycler with the heated lid set to 80°C using the following conditions: 25°C for 10min, 42°C for 50min, 70°C for 15min, hold at 4°C. Plate was then removed, briefly centrifuged, and second strand cDNA synthesis reaction began immediately. Next, 60μl second strand mastermix (composed of nuclease free water, NEBNext second strand synthesis buffer, and NEBNext second strand synthesis enzyme mix) was added to the first strand synthesis reaction. The plate was sealed and placed in thermal  107  cycler for 60min at 16°C. AMPure XP beads were resuspended and added to the second strand synthesis reaction to purify the double-stranded cDNA. The supernatant was subsequently discarded and washed in 80% ethanol, airdried, and the DNA target was eluted from the beads by adding 53μl 0.1x TE buffer to the beads. The end prep mastermix was prepared by combining NEBNext Ultra II end prep reaction buffer and NEBNext Ultra II end prep enzyme mix, and added to the purified cDNA. The sealed plate was then placed in a thermal cycler at 20°C for 30min, and 65°C for 30min, and removed when the incubation reached 4°C.  Following adaptor ligation and purification using AMPure XP beads, the PCR library was enriched by adding 25μl of NEBNext Q5 Hot start HiFi PCR master mix and 10μl of pre-assigned index primer to each well and incubation. The thermal cycler lid was heated to 105°C, and samples went through: 1 cycle of 98°C for 30sec, 98°C for 10sec, 13 cycles of 65°C for 75sec, 1 cycle of 65°C for 5min, and held at 4°C. The PCR reaction was then purified using Agencourt AMPure XP beads. All resulting libraries were quantified using the Qubit HS dsDNA kit and qualified on the appropriate 1000bp ScreenTape according to concentration.  Paired end mRNA sequencing was performed using the NextSeq 500 High Output Kit (Blizard Genome Centre) with 20M reads and a read length of 75bp per sample.