GSM6703011: RSB217-171 [Yohimbine HCl]; Homo sapiens; miRNA-Seq
On day 20, four 10x photos were taken per well automatically using the ImageXpress Micro Confocal microscope (Molecular Devices). Then, the media in the plates was removed with approximately 20 μL media left, using a 24-channel stainless steel manifold (Drummond #3-000-101) linked with a vacuum at a low speed. With the help of Multidrop™ Combi Reagent Dispenser (Thermo Scientific) and the standard cassette (speed: low), 250 μL ice-cold DPBS (Wisent) was added per well. The DPBS was removed with approximately 20 μL liquid left, using another 24-channel stainless steel manifold linked with the vacuum at a low speed. The residual DPBS was completely removed using a mechanic 12-channel pipette. Next, 45 μL lysing solution was added per well using another Multidrop™ Combi Reagent Dispenser with the small cassette (speed: low), where the lysing solution is composed of single-cell lysis buffer (Takara #635013): 1x RNase Inhibitor Murine (NEB): nuclease-free water (Exiqon) = 19:1:190. Thorough lysis was achieved by shaking the plates on a shaker for 5 min at room temperature. The lysis samples were transferred from four 96-well plates to each 384-square-well plate using the 96-well module-coupled VPrep liquid handler (Agilent) with 30 μL tips (twice without changing tips). After sealing, the 384-square-well plates were spun down at 4000 rpm for 5 min, and 10 μL supernatant was aliquoted to a 384-well plate (Eppendorf) using the 384-well module-coupled VPrep liquid handler. The plates were finally sealed with the PlateLoc Heat Sealer (Agilent) and stored at -20oC. To avoid RNA purification, we used RealSeq-T technology (RealSeq Biosciences) following manufacturer recommendations. In summary, cell lysates were incubated at 70C for 5 minutes on RealSeq hybridization buffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1mM DTT, pH 7.9) with 1x RealSeq biotinylated DNA probes to target all miRNAs in miRbase 21. After 2 hours of incubation at 37C, 10 μL of RealSeq Beads were added, and miRNAs were captured using a 384-well Magnet Plate (Alpaqua, MA). Following three washes with RealSeq Wash buffer, miRNA was eluted from beads in 10uL of RNase-free water. All the miRNA elusion was input to prepare sequencing libraries with RealSeq-Biofluids following manufacturer instructions (RealSeq Biosciences). In summary, a single adapter and circularization approach was used. Libraries were barcoded with dual indexes and sequenced with a NextSeq 550 (Illumina, CA).