GSM7401857: CS13_Gonadal_Epithelium_Proximal_Rep2 (GEP_2); Homo sapiens; RNA-Seq
The previously published LCM-Seq protocol (Nichterwitz, et al. 2016) was used for RNA-Seq library preparation from LCM material in lysis buffer. LCM caps were vortexed for 15 s and spun in a tabletop centrifuge (8000 g) for 5 min. 5 μL lysate was added to 2 μL 10mM dNTP mix (Thermo Fisher) and 1 μL 10 μM oligodT (5′-AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′, (IDT)). This was vortexed briefly and spun in a microcentrifuge for 30 s (700 g) then incubated at 72°C for 3 min and immediately snap cooled on ice. To each reaction, 2 μl SSRTIV 5 × buffer, 0.5 μl 100 mM DTT, 0.5 μl 200 U μl−1 SSRTIV (all Thermo Fisher), 2 μl 5 M betaine (Sigma-Aldrich), 0.1 μl 1 M MgCl2 (Sigma-Aldrich), 0.25 μl 40 U μl−1 RNase inhibitor (Takara) and 0.1 μl 100 μM TSO-LNA-oligo 5′- AAGCAGTGGTATCAACGCAGAGTACATrGrG+G −3′, Exiqon) was added. The reverse transcription reaction was performed in a thermal heat cycler with the following conditions; 90min 42°C, 10 cycles of (2 min 50°C, 2 min 42°C) and 15 s 70°C. For the amplification reaction 12.5 μl 2X KAPA HiFI Hotstart Mix (KAPA Biosystems), 0.2 μl 10 μM ISPCR primers (IDT, 5′ - AAG CAG TGG TAT CAA CGC AGA GT –