GSM5665328: Ovx beta hCG transgenic Progesterone treated-1; Mus musculus; RNA-Seq
Tissue samples were homogenized in TOMY Microsmash in presence of liquid nitrogen. TRIZOL reagent (Ambion#15596018) was added and incubated at room temperature for 5 minute, lysate was centrifuged at 13500 rpm for 10 minutes to remove debris followed by phase separation by Chloroform. Aqueous phase was separated and RNA was precipitated with Ethanol. Rest of the protocol was followed as per manufacturer's guidelines including on column DNase treatment. RNA was eluted in Nuclease free water (Ambion, USA). The quantification and quality of the genomic DNA was assessed using Nanodrop2000 (Thermo Scientific, USA) , Qubit (Thermo Scientific, USA) and Bioanalyzer 2100 (Agilent, USA). RNA sequencing libraries were prepared with Illumina-compatible NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. 500 ng of Qubit quantified total RNA was taken for mRNA isolation, fragmentation and priming. Fragmented and primed mRNA was further subjected to first strand CDNA synthesis followed by second strand synthesis. The double stranded cDNA was purified using HighPrep Beads (Magbio, Cat # AC-60050). Purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext® Ultra™ II Directional RNA Library Prep protocol followed by second strand excision using USER enzyme at 37 ˚C for 15mins. Adapter ligated cDNA was purified using HighPrep Beads and was subjected to 11 cycles for Indexing- (98˚C for 30 sec, cycling (98˚C for 10sec, 65˚C for 75sec) and 65˚C for 5min to enrich the adapter-ligated fragments. Final PCR product (sequencing library) was purified with HighPrep Beads, followed by library quality control check. Illumina-compatible sequencing library was quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA).