GSM8958364: SNCAdupl_rep3; Homo sapiens; RNA-Seq
Approximately ~200,000 iPSC-derived neurons were stored in TRIzol. Total RNA was extracted from homogenates and cells using the Direct-zol RNA MicroPrep Kit. Total RNA was extracted from homogenates and cells using the Direct-zol RNA MicroPrep Kit (Zymo Research R2052) and stored at -80°C until RNA-seq library preparation. mRNAs were enriched by incubation with Oligo d(T) Magnetic beads (NEB, S1419S) in 2× DTBB buffer (20 mM Tris-HCl pH 7.5, 1 M LiCl, 2mM EDTA, 1% lithium dodecyl sulfate, 0.1% Triton X-100) at 65°C for 2 minutes and were incubated at room temperature while rotating for 15 minutes. Beads were washed 1× with RNA Wash Buffer 1 (10mM TrisHCl pH 7.5, 0.15M LiCl, 1mM EDTA, 0,1% lithium dodecyl sulfate 0.1% Triton X-100) and 1× with RNA Wash Buffer 3 (10mM Tris-HCl pH 7.5, 0.15 M NaCl, 1mM EDTA) before elution in RNA Elution Buffer (10mM Tris-HCl pH 7.5, 1mM EDTA) at 80°C for 2 minutes. PolyA selection was performed a second time, and samples were washed 1× with Wash Buffer 1, 1× with Wash Buffer 3, and 1× with 1× SuperScript III first-strand buffer. Beads were resuspended in 10 µL 2× SuperScript III buffer plus 10 mM DTT, and RNA was fragmented at 94°C for 9 minutes and immediately chilled on ice before the next step. For first-strand synthesis, 10 µL of fragmented mRNA, 0.5 µL Random primers (50 µM) (Thermo Fisher), 0.5 µL SUPERase-In (Ambion), 1µL dNTPs (10mM), and 1µL of DTT (10mM) were heated for 50°C for one minute. At the end of incubation, 5.8 µL of water, 1 µL of DTT (100mM), 0.1 µL Actinomycin D (2µg/uL), 0.2uL of 1% Tween-20 (Sigma) and 0.5µL of SuperScript III (Thermo Fisher Scientific) were added and incubated in a PCR machine using the following conditions: 25°C for 1 minute, 50°C for 50 minutes, and a 4°C hold. The product was then purified with RNAClean XP beads (Beckman Coulter) according to the manufacturer's instruction and eluted with 10 µL nuclease-free wate. The RNA/cDNA double-stranded hybrid was then added to 1.5 µL Blue Buffer (Enzymatics), 1.1 µL of dUTP mix (10mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 µL RNase H (5U/uL), 1.05 µL of water, 1µL of DNA Polymerase I (Enzymatics) and 0.15 µL of 1% Tween-20. The mixture was incubated at 16°C overnight. The following day, the dUTP marked dsDNA was purified using 28 µL of SpeedBeads (GE Healthcare), diluted with 20% PEG8000, 2.5M NaCl to a final concentration of 13% PEG, eluted with 40 µL elution buffer (DNA elution buffer from Zymo ChIP Clean and Concentrator Kit). The purified dsDNA underwent end repair by blunting, A-tailing, and adaptor ligation as previously described (79) using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR amplified for 16 cycles, size for 200-500bp size range, quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and sequenced on a NovaSeq 6000 for 51 cycles according to the manufacturer's instructions.