GSM8240251: fatbody of cricket, group A, 1st injection = PBS, rep1; Gryllus bimaculatus; RNA-Seq
To perform RNA sequencing, we extracted fatbodies from group A, B, A-1, and B-1. We selected 10 crickets from each group and prepared them in one tube for each group. Since each group was repeated 3 times, fatbodies were extracted from a total of 30 crickets and prepared in 3 tubes per group (12 tubes in total). Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, USA) (all operations were performed using sterile spaces and tolls to minimize contamination). To briefly summarize the procedures, 100mg of fat bodies were mixed with 1ml of TRIzol reagent (Thermo Fisher Scientific, Waltham, USA). Plastic beads of 0.6 mm, 2 mm, and 7 mm were added to the mixture, and it was homogenized for 10 minutes with a vortex (DAIHAN Ltd., Korea). The sample were centrifuged at 14,000 rpm for 3 min at 4℃ and 1mL of supernatant was extracted. The extracted supernatant was mixed with 200 µL chloroform and reacted at 4℃ for 20 min (Thermo Fisher Scientific, Waltham, USA). The sample were centrifuged at 14,000 rpm for 3 min at 4℃ for 15min and then the supernatant (~600 µL) was loaded into a gDNA elimination column to remove gDNA (Thermo Fisher Scientific, Waltham, USA). The sample was centrifuged at 14,000 rpm for 1 min at 4℃ and then total RNA was precipitated by adding 600 µL of cold isopropanol/70% ethanol (maintained at -20℃, Sigma-Aldrich, USA). The sample was centrifuged at 14,000 rpm for 1min at 4℃ using RNeasy Plus Mini column, mixed with ~700 µL of RW1 buffer (10mM Tris-Hcl, 1mM EDTA, 50mM NaCl, 0.5% Tween-20, pH 8.5, Sigma-Aldrich, USA), and centrifuged at 14,000rpm for 1min. Finally, the sample was mixed with 500 RPE buffer (10mM Tris-Hcl, 80% ethanol, 0.5mM EDTA pH 7.5), centrifuged, and total RNA was extracted by placing ~50 µL RNeasy-free water in a new sterilized tube (Sigma-Aldrich, USA). The cDNA library was generated from the total RNA, and paired-end sequencing was performed using the Illumina RNA-seq technology (Macrogen Ltd, Korea).