title: RNA-Seq data for Ffuji and Cured-Ffuji
design: For RNA sequencing (RNA-seq), 0.1-1.0 g of Protein-free, total RNA (RIN>7) was used for library construction using the NEBNext UltraTM II RNA Library Prep Kit (Cat #E7770), the NEBNext Poly(A) mRNA Magnetic Isolation Module (Cat#E7490), and the NEBNext Multiplex Oligos (Cat# E7335S). Briefly, RNA was fragmented in a solution containing divalent cations, with incubation at 94C. This step was followed by first strand cDNA synthesis using reverse transcriptase and oligo-dT primers.  Synthesis of double strand cDNA was done using the second-strand master mix provided in the kit, followed by end-repair and dA-tailing.  At this point, Illumina adaptors were ligated to the sample.  Finally, the library was enriched (and barcoded) by 5-10 cycles of amplification, and purified by AMPure beads (Beckman Coulter, catalog # A63881).  Quantitation was done by fluorescence (QUBIT, ThermoFisher), and average library size (bp) estimated on the Agilent TapeStation. A yield of approximately 100 ng of library was obtained (~600 bp long) with a 400-100 bp fragment size distribution. The libraries were subsequently normalized and pooled at various proportions based on desired output per sample, for sequencing on a single Illumina MiSeq, 2x300 cycles run (v3).
identifiers.namespace: SUB6265356
identifiers.id: Ffuji_343