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Library Preparation: Libraries were prepared using the Universal Plus mRNA-Seq with (NuGEN Cat# 0520-24). a. Poly(A) Selection: 8-30 ng of RNA samples (in a volume of 50 μl) were mixed with Oligo (dT) Beads and incubated at 65oC for 5 minutes, mixed, then at room temperature for 10 mins. The supernatant was discarded and the beads were washed with wash buffer. Captured polyadenylated RNA was eluted using Elution buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up. b. RNA Fragmentation: Beads were mixed with 20 μl Fragmentation Buffer and incubated for 8 minutes at 94oC. 20 μl of supernatant was removed from the beads and proceeded immediately to First Strand cDNA Synthesis. c. First Strand cDNA Synthesis: 5 μl of First Strand Master Mix was added to each sample and placed on a thermal cycler to run preprogrammed thermal conditions. d. Second Strand cDNA Synthesis: 50 μl Second Strand Master Mix was added to each sample, mixed well and incubated in a thermal cycler at 16oC for one hour. e. cDNA Purification: cDNA was purified using Agencourt AMPure XP Beads (1.8 volumes), eluted in 10 μl of H2O, and stored at -20oC. f. End Repair: End Repair Mix was added to the purified samples from step e and the samples were incubated in a preprogrammed thermal cycler. g. Adaptor Ligation: Samples were mixed with 3 μl adaptor mix, then 12 μl Ligation Mix, and incubated in a thermal cycler at 25oC for 30 minutes. Sample and Barcode Information: Samples were barcoded with NuGEN Unique Dual Indexes. Strand Selection: 70 μl of Strand Selection Master Mix was added to 30 μl of each sample, incubated in a pre-heated thermal cycler at 72oC for 10 minutes. Strand Selection Purification: Strand selected cDNA was purified using Agencourt AMPure XP Beads (0.8 volumes), eluted in 15 μl of H2O, and stored at - 20oC. j. Library Amplification: 76.5 μl of Library Amplification Master Mix was added to 13.5 μl of each sample and incubated in a preprogrammed thermal cycler, for 13- 16 cycles, which was pre-optimized with qPCR. k. Amplified Library Purification: Amplified libraries were purified using 90 μl Agencourt AMPure XP Beads (1.0 volumes). 25 μl of eluted libraries were collected and stored at -20oC. l. Validate Library: The concentration of libraries were measured by Qubit dsDNA HS Assay Kit (Invitrogen Q32851). Libraries were diluted and normalized to the optimal range for Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit (Agilent Technologies, Cat# 5067-4626). m. Normalize and Pool Libraries: Same amount of libraries were pooled based on the molar concentration from Bioanalyzer measurements. Library Denaturing and Diluting for MiSeq Nano 300: Pooled library was run on MiSeq to test quantity and quality, using the MiSeq Reagent Nano Kit V2 300 cycles (Illumina, Cat. No. MS-103-1001). Library and PhiX control (Illumina, Cat. No. FC-110-3001) were denatured and diluted using the standard normalization method following manufacturer's directions, to a final concentration of 6pM. Equal volume of library and PhiX were combined and sequenced on Illumina MiSeq. (https://basespace.illumina.com/run/196126950/JY_Xiaoling07072020_test) 3. Library Denaturing and Diluting for Nextseq 500: Library and PhiX were denatured and diluted using the standard normalization method following manufacturer's directions. The loading of library pool was 1.3 ml at 1.8 pM, with 1% PhIX spike in. Sequencing Run: Sequencing was performed on the University of Louisville Brown Cancer Center Genomics Core Illumina NextSeq 500 using the NextSeq 500/550 75 cycle High Output Kit v2.5 (20024906). |
