PacBio Iso-Seq of human lung adenocarcinoma cell line post treatment with KRAS morpholino
NEBNextSingle Cell/Low Input cDNA Synthesis: Briefly, 300 ng of total RNA was reverse transcribed usingNEBNextSingle Cell/Low Input Kit for cDNA synthesis following the steps outlined within Iso-Seq Express Template Procedure & Checklist ((PN 101-763-800 Version 02 (October 2019)). Theresulting cDNA waspurifiedand size selected at >1.0 kb utilizing 1.0xProNexBeads (Promega, Madison, WI) with a final elution of 17 l in Buffer EB (Qiagen, Hilden, Germany). Subsequent PCRamplification usedTakara PrimeStar GXLDNA polymerase(2.50 Units total) withNEBNextSingle Cell (catalog #E6421S) and ISO-Seq Express (PN #101-737-500) cDNA primers;primer binding site incorporation occurred during first-strand cDNA synthesis. Sample cDNA amplification occurred in duplicate 50 l PCR reactions with each containing 8 l purified first-strand cDNA template, 1xPrimeSTARGXL Buffer, 0.1 mM dNTPs, 1.25 UnitsPrimeSTARGXL DNA Polymerase (1.25U / l),1 lNEBNextSingle Cell and Iso-Seq Express cDNA PCR Primers.PCR cycling conditions were as followed: 30 seconds at 98oC, 15 cycles (10 seconds at 98oC, 15 seconds at 65oC, 10 minutes at 68oC) and 5 minutes at 68oC. cDNA Size Fractionation: Combining duplicatePCR reactionspost-amplificationallowed for >2kbsizeselection using 0.4X and 0.4/0.6Xratios of SPRIselectparamagnetic beads (Beckman Coulter, Brea, CA). Supernatant from the 0.4X sizing was added to 20 lSPRIselectbeads yielding a double-sidedsize-selected product between 0.5  2 kb. Bead-associated cDNA was eluted and concentrated in 12 l Buffer EB. Pooling the fractionated size-selected cDNA (final yield of 500 ng) at a1:1 molar ratio of the >2kb (0.4x) and 0.5-2kb (0.4/0.6x) fractions allowed for equal representation of the larger-sized molecules during PacBio SMRTcell loading and sequencing. SMRTbellTemplate Prep and Sequel II Sequencing: Single molecule real-time (SMRT)belllibrarypreparation of the pooled cDNA fractions used theSMRTbellExpress Template Prep Kit 2.0 according to PacBio Iso-Seq protocol (PN 101-763-800 Version 02 (October 2019)). A five-fold dilution derived fromthefinalSMRTbelllibrary was used for assessing concentration and average library size by Qubit dsDNA HS and 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent), respectively. Volumetric calculations were done on a per-sample basis using SMRT Link Sample Setup for v4 primer and polymerase binding conditions dependent upon initialSMRTbelllibrary molarity.SMRTbelllibraries werecomplexed with Sequel II Binding Kit 2.0,purified with 1.0x ProNexbeads,and sequenced at 60pMon-plate loading concentration. Samplesweresequenced on a single 8M SMRTCell on the PacBio Sequel IIe platform using v2.0chemistries.Sequencingincludeda2-hour pre-extensionand a24-hour movie collection.
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