Confluent human primary thyroid cultures were exposed to human IFNα (5,000 U/ml) for 0, 12 or 24 hours, followed by RNA isolation.
Human thyroid primary cells were prepared from fresh, non-cancerous thyroid tissue adjacent to thyroid tumors that were removed at surgery. Tissue was minced and incubated in 200 U/ml of collagenase solution for 1 hour at 37°C. Cells were harvested and cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) (P0). Cells were passaged at 1:2 dilution and cultured until confluent.
According to the Illumina mRNA Sequencing Sample Preparation Guide. Briefly, 2ug total RNA was hybridized to poly-dT magnetic beads to capture polyA mRNA molecules. Chemical fragmentation, first-strand synthesis with RTase, randomers second-strand synthesis. Then, end repair, 3' A overhang, multiplex adapter ligation (from Illumina indexing kit), size selection by electrophoresis and cut at 200bp. 18 cycles of PCR with a primer bearing an indexing barcode.
Illumina Genome Analyzer IIx
RNA-seq of mouse thyroid tissue from wild type littermates.
Single reads split by barcode into individual files using PERL script. The reads with good quality were aligned to reference sequence (RefSeq) databases of human (UCSC hg18) or mouse (UCSC mm9) genomes, RefSeq exons, splicing junctions and contamination databases including ribosome and mitochondria sequences using the BWA alignment algorithm, and the alignment files in SAM format were generated. After filtering reads mapped to contamination databases, the reads that have one or no mismatch and are uniquely aligned to each exon and splicing-junction sites were extracted and then counted. The read count for each RefSeq transcript was calculated by combining the counts for exons and splicing junctions of the corresponding transcript. The read count at exon, splice-junction, transcript and gene levels were summarized and normalized to relative abundance in Fragments Per Kilobase of exon model per Million (FPKM) in order to compare transcription level among samples.