GSM9116399: stomach female control rep5; Rattus norvegicus; RNA-Seq
A 50% (v/v) solution of Antifoam A (cat. no. A5633; Sigma, St. Louis, MO) was prepared in propylene glycol (cat. no. W294004; Sigma St. Louis, MO). The solution was vortexed prior to use. Homogenization buffer was prepared by adding 10 µl of β-mercaptoethanol (cat no. 0482, VWR, Radnor, PA) and 60 µl antifoam solution per milliliter of Buffer RLT Plus (Qiagen, Germantown, MD). Prior to homogenization, large tissue samples were broken into smaller pieces using a mortar and pestle cooled with liquid nitrogen (LN2). Several of the resulting pieces (each approximately 3–5 mm) were then pulverized together to produce the final sample. For homogenization, 400 µl of buffer was added to a 3 ml, flat-bottom polystyrene tube (Globe Scientific, Mahwah, NJ). The tube was placed into a CoolRack CF15 (Corning Inc., Corning, NY) cooled with LN2. A cooled 30 µl plastic scoop was used to transfer pulverized tissue to the tube. The target mass of tissue was 50 µg, although the actual mass varied due to the need to work quickly and differences in the sizes of the pulverized samples. The tube was placed on ice, and 600 µl of ice-cold homogenization buffer was added to the sample. The sample was homogenized using a model 985-370 TissueTearor rotor-stator homogenizer with a 7 mm probe (BioSpec Products, Bartlesville, OK) until no remnants of intact tissue were visible. If the homogenate appeared significantly darker than other samples, suggesting an excess of tissue, additional buffer was added until the color of the sample was similar to that of the other samples. Homogenates were stored at -80 °C until used for RNA isolation. The homogenizer was rinsed between samples within a treatment by running it at the highest speed for 30 s in three changes of deionized water. Between treatments, the rotor and stator were disassembled and cleaned with a polyester test tube brush before rinsing in water. For RNA isolation, homogenized samples were removed from the freezer and placed on ice to thaw. The samples were vortexed, and 100 µl of the homogenate was transferred to the well of a 96-well round bottom plate (Caplugs Evergreen, Buffalo, NY). RNA was isolated using the MagMAX-96 Total RNA Isolation Kit (Fisher Scientific, Hampton, NH) according to the manufacturer's protocol, except for the substitution of RLT Plus buffer for the lysis/binding solution as described above. Isolated RNA was quantified on a Synergy HTX plate reader using a Take3 microvolume plate (Agilent, Santa Clara, CA). The integrity of the RNA was analyzed on an Agilent TapeStation using RNA Screen Tapes and reagents (Agilent, Santa Clara, CA). Concentrations of samples ranged from 75 to 470 ng/µl. RNA integrity numbers (RIN) ranged from 8.0 to 10.0; nearly 90% of samples had a RIN higher than 9.0. Isolated RNA was next used to prepare RNA-Seq libraries. Samples were blocked so that about eight samples of each tissue and treatment combination were included in one of two plates. RNA was normalized to 25 ng/uL with nuclease-free water (Ambion, USA). 250 ng total RNA (10 uL of normalized RNA) was input into mRNA purification with magnetic beads using a PolyA Selection Module (Lexogen, USA). Purified mRNA was then used directly in CORALL mRNA-Seq V2 library preparation with set A unique dual indexes (UDIs). Each batch was checked by qPCR to determine the appropriate number of PCR cycles for library amplification with Add-On PCR V2 kit (Lexogen, USA), SYBR Green (ThermoFisher, USA), and a QuantStudio 6 Flex real-time PCR instrument (ThermoFisher, USA). After final PCR amplification, libraries were quantified with Quant-iT PicoGreen (ThermoFisher, USA). The distribution of library size was checked by TapeStation D1000 assay (Agilent, USA). The average library size was 508 – 517 bp. Samples below 1 ng/uL were re-amplified.