Instrument: Illumina HiScanSQ
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Approximately 30 mg of endometrial tissue were ground in liquid nitrogen using a stainless steel apparatus and immediately mixed with buffer RLT from the RNeasy Mini columns kit (Qiagen, São Paulo, SP, Brazil), as per manufacturer’s instructions. To maximize lysis, tissue suspension was passed at least ten times through a 21 G needle, and centrifuged at 13,000 x g for 3 minutes for removal of debris, prior to supernatant loading and processing in RNeasy columns. Columns were eluted with 40 l of RNase free water, and elution was repeated using the same 40 l initially used to increase RNA concentration. Concentration of total RNA on extracts was measured by a spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, USA). Prior to reverse-transcription, 1 g of total RNA was treated with DNase I (Life Technologies, São Paulo, SP, Brazil) for 15 minutes at room temperature in a 10 l reaction, followed by addition of 1l of EDTA (25mM) and heating at 65C for 10 minutes to inactivate DNase I. DNase I treatment was immediately followed by reverse-transcription (High Capacity cDNA Reverse Transcription Kit, Life Technologies) according to manufacturer’s instructions. Briefly, 9 l of master mix containing RT buffer, dNTP mix, random primers, RNase inhibitor and reverse transcriptase were added to the 11 l of DNase I treatment reaction. Immediately, samples were incubated at 25 C for 10 minutes, followed by incubation at 37 C for 2 hours and reverse-transcriptase inactivation at 85C for 5 minutes and storage at -20C. RNA libraries were prepared for sequencing using standard Illumina protocols