title: GSM9246286: CGND-HRA-02965; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA extraction from fresh frozen tissue or cells – starting material is homogenized in TRIzol reagent using a series of needles for trituration. The homogenized sample undergoes phase separation with the addition of chloroform,  the upper aqueous phase of the mixture then follows the RNeasy Mini procedure (Qiagen, 74106). A series of washes will purify the final eluted total RNA. The RNeasy Mini portion of the protocol is processed on the QIAcube Connect automated platform (Qiagen, 9002864) for higher throughput. RNA sequencing libraries are prepared using the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) (Roche 07962304001) in accordance with the manufacturer's instructions. Samples are normalized to 500ng and undergo rRNA depletion through oligo hybridization and RNase H treatment. DNase treatment removes DNA oligos. Samples are fragmented to 350bp inserts and undergo cDNA generation. Samples undergo A-tailing and are ligated to Illumina sequencing adapters and amplified. Final libraries are quantified using the Qubit Fluorometer (Life Technologies) or Spectramax M2 (Molecular Devices) and Fragment Analyzer (Agilent). Libraries are sequenced on an Illumina sequencer.