FGF19 signaling controls liver size in humanized chimeras
Sequencing libraries were constructed using the Illumina TruSeq RNAseq kit. Briefly, poly(A)+ RNA was isolated from total RNA using oligo-dT coated magnetic beads. The recovered RNA was then chemically fragmented. First strand cDNA was generated using random hexamers as primers for reverse transcriptase. Following second strand synthesis, the ends of the double stranded fragments were repaired and then a single “A” nucleotide was added to each end. Illumina adaptors were ligated to the cDNAs. Limited round PCR was used to amplify the material to yield the final library. Library concentration was determined using real time PCR with primers complementary to the Illumina adaptors. Sample libraries were diluted and applied to an Illumina paired end flow cell at a concentration appropriate to generate about 180 million reads per lane. All libraries were prepared with indexing barcodes to permit multiplex runs. 50 cycle single read sequencing were used to generate base call files. Illumina’s CASAVA package was used to assemble the reads into standard fastq formatted data.