GSM6998409: Library_5_dilut100_rep_1; synthetic construct; RNA-Seq
Synthetic RNA in water for the synthetic libraries.  Jejunal RNA was extracted using the TRI Reagent method (Molecular Research Center; Cincinnati, OH). RNA samples were stored at -80o C until needed for Illumina sequencing. Library construction followed Nanopore's instruction for the SQKPCS109  kit, except for samples that included a poly-adenylation step. Those samples were subjected to a 15 PAP reaction, followed by cold inactivation of the PAP enzyme. Then the entire product was used as input to the SQKPCS109  protocol without an intermediate cleanup. Biological samples were subjected to a PAP step, except for those designated as "R10" : they were processed with the unmodified Nanopore protocol. Number of PCR cycles: biological samples subjected to the unmodified Nanopore protocol :10, Biological samples subjected to PALS-NS : 18, , Libraries 1-4 & Libraries 1-5 (up to 10 fold dilution) : 15, Library 5 100fold diluted 19, Library 5 1000 fold diluted 22 RNAseq with or without an optional polyadenylation step. Synthetic libraries were made under conditions of sham polyadenylation, in which all the re-agents for the reaction except the PAP enzyme were included in the mix. These sham polyadenylated samples were subjected to the same conditions (incubation and cold treatment) as their PAP counterparts.