GSM7401852: CS13_Gonadal_Epithelium_Distal_Rep1 (GED_1); 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 – 3′) and 2.3 μl nuclease-free H2O (Invitrogen) was added to each reaction. This was heat cycled as follows: 3 min 98°C, 18 cycles of (20 s 98°C, 15 s 67°C, 6 min 72°C) and 5 min 72°C. After bead purification using AMPure XP beads (Beckman Coulter), the concentration of the cDNA library was measured with an Agilent 2200 TapeStation using the High Sensitvity DNA 5000 kit (Agilent). 1 ng of cDNA from this reaction was amplified and barcoded using the Nextera XT DNA sample preparation kit and Nextera XT index kit (Illumina) following the manufacturer's protocol. The libraries were purified again using AMPure XP beads, analyzed on the Tapestation 2200 using High Sensitivity DNA 100 Kit and quantified using the Qubit fluorometer and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).