GSM7099816: KFs, MDI medium plus GO6983, 8 Days; Homo sapiens; RNA-Seq
For small molecule screening, 128 compounds were collected based on functions in the signaling pathway and cell fate transition. KFs were seeded at a density of 0.5 × 104 cells per well of a 96-well plate containing KF medium. The next day, the medium was changed to MDI medium (DMEM/high glucose, 1% ITS, 0.5mM isobutylmethylxanthine, 1 μM dexamethasone) plus 2 μM compounds. After 4 days, DMEM/high glucose with 1% ITS was added to the culture for another 4 days. The negative control was KF in KF medium for 8 days. The positive control was KF in AD medium for 4 days and then AM medium for another 4 days. Cells were harvested on day 8 for PHDs-seq library preparation. Samples with compound treatments in a 96 well-plate were washed with pre-warmed PBS. 60 μl of lysis buffer containing 50mM Tris-HCl pH8.0, 75mM KCl, 6% Ficoll PM-400, 0.15% TritonX-100, and 0.5U/μl RNaseOUT (RNaseOut should be added before use) were added to each well. The 96-well plate was then incubated on a shaker at 900 rpm for 15 minutes. 4.286 μl of cell lysate were transferred to a new 96 well PCR plate. 0.714 μl reverse transcription mix containing 40mM MgCl2, 4μM strand switching primer (TSO), 0.1mM dNTP, 8U/μl Maxima reverse transcriptase, 1x ERCC, and RNaseOUT (RNaseOut should be added before use) were added to each well, mixed and slightly centrifuged for several seconds to collect all reactants on the bottom. The 96-well PCR plate was incubated at 42°C for 2 hours with a hot lid at 105°C. 6μl of hybridization-ligation mix (0.005 μM probe mix, 1U/μl Taq DNA ligase, 1x Taq DNA ligation buffer) was added to the RT products, mixed gently, and incubated at 60°C for 60 minutes with a hot lid of 105°C. After hybridization and ligation, the products were enriched with 15 ul of enrichment mixture (2 μl Dynabeads MyOne Carboxylic Acid beads plus 13 μl capture buffer containing 30% PEG-600, 2 M, NaCl, 5 mM Tris-HCl pH7.5, 10 mM EDTA, and 0.02% Tween-20). Next, 20 μl of PCR mixture (0.25 μM PHD-seq well barcode, 0.5 mM PHD-seq plate barcode, 1 x Vazyme LAmp Master Mix) were added to the enriched hybridization-ligation products, mixed, and incubated at 94°C for 5min, followed by two cycles of 95°C for 20 s, 57°C for 30 s and 72°C for 20 s. In addition, 10 cycles of 94°C for 20 s, 65°C for 30 s, and 72°C for 20 s with a final extension at 72°C for 60 s. The PCR products were pooled together into several 1.5 ml tubes which were placed on a magnet to remove the beads. The supernatant was purified with DNA Clean & Concentrator-100 column (Zymo Research, D4029) and eluted with 150 µl of ddH2O. The library was size-selected using AMPure XP beads. 150 µl of beads were added to 150 µl of the purified PCR product, incubated for 5 min, and captured by a magnet for 3 min after which the supernatant was discarded. The beads were eluted in 25 µl of ddH2O and incubated for 1 min at room temperature. After 1 min of incubation on the magnet, the supernatant was transferred to a clean 1.5 ml tube. The 208 bp library was visualized using agarose gel electrophoresis and quantified using a Qubit™ 4 NGS Starter Kit with WiFi (Q33240).