GSM9128015: Keloid fibroblasts，KF medium plus compounds, rep2; Homo sapiens; RNA-Seq
DRUG-seq2: Samples with compound treatments in a 96 well-plate were washed with pre-warmed PBS. 60 μL lysis buffer containing 50 mM Tris-HCl pH8.0, 75 mM 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 1,000 rpm for 15 minutes. The lysates were then transferred to a new 96-well PCR plate.10 μL reverse transcription mix containing 40 mM MgCl2, 4 μM strand switching primer (TSO), 0.1 mM dNTP, 8 U/μL Maxima reverse transcriptase, 0.4 μL 1:100 diluted ERCC, and RNaseOUT (RNaseOut should be added before use) were added to each well, mixed, and lightly centrifuged for several seconds to collect all reactants at the bottom of well. A 96-well PCR plate was then incubated at 42°C for 2 hours with a hot lid of 105°C. After RT-PCR, products from a 96-well plate were pooled into a clean 50 mL tube and then twice purified according to the manufacturer's instructions of DNA clean & concentrator-100 kit (ZYMO Research, D4029) and Agecourt RNA Clean XP beads (BECKMAN COULTER, A63987), respectively. The purified products were dissolved in 32 μL H2O. Next, 4 μL exonuclease I (NEB, M0293S) and 4 μL exonuclease I buffer were added to the above solution and then incubated in 37°C for 30 min, followed by 85°C for 15 min to digest the residual primers. Products from the digestion were directly amplified with Kapa HIFI PCR ReadyMix (Roche, KK2602) and 1 μM DRUG-seq2 pre-amplification primer via the PCR cycling conditions: 96°C for 1 min, 5 cycles of 98°C for 20 s, 58°C for 4 min, and 72°C for 6 min; 10 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 6 min, with a final extension at 72°C for 10 min. PCR products were purified and collected with 25 μL H2O according to the manufacturer's instructions (Agecourt RNA Clean XP beads; BECKMAN COULTER, A63987). Pre-amplification products were quantified via Qubit 4.0 (Thermofisher, Q33240) and then analysed on an Agilent Fragment Analyzer 5400. The result of fragment distribution should be a single peak at around 1,500~3,000bp with slight degradation. Qualified samples were then processed to obtain the pre-library that included fragmentation with Tn5 and 3'-end enrichment with 5 μM DRUG-seq2_P5_PCR primer and 5 μM Plastech--seq indexing primer (TruePrepTM DNA Library Prep Kit V2 for Illumina (Vazyme, TD502). The PCR procedure included: 72°C for 3 min, 98°C for 30 s, 15 cycles of 98°C for 15s, 60°C for 30s, and 72°C for 3 min, with a final extension of 72°C for 5 min. The pre-library was used to select 250-450 bp products with Agencourt AMPure XP beads (BECKMAN COULTER, A63880). Briefly, 50 μL pre-library was transferred to a 1.5 mL tube and mixed with 35 μL AMPure XP beads until the solution appeared homogeneous. After incubating for 5 min at room temperature, the 1.5 mL tube was placed on the magnet stand for 5 min until the solution was clear and the beads were collected at one corner of the tube. The supernatant was aspirated and discarded. The beads were washed twice with 500 μL fresh 80% ethanol for 30s at room temperature. During the washing, the beads remained on the magnet strand to avoid disturbing the separated magnetic beads. The tube containing the magnetic beads was then air-dried for 5 min with the cap open. After that, the washed and air-dried beads were eluted by 26.5 μL nuclease free water. The tube was incubated at room temperature for 5 min and then placed on the magnet stand for 5 min to separate the beads and solution. Finally, 25 μl solution was collected into a new 1.5 mL tube without disturbing the beads. This solution (library) was quantified via Qubit 4.0 (Thermofisher, Q33240), and then analysed on the Agilent Fragment Analyzer 5400. The qualified library was sequenced on the Illumina Hiseq X-ten platform.