Allprep mini kit

For sample preparation from low cell number and BT474 small total RNA inputs (10ng-10pg) the Ovation Single Cell RNA-Seq System (NuGEN, San Carlos, CA) was used. For the BT474 bulk cells and PB cell total RNA isolation was performed using the AllPrep Mini Kit and QIAamp RNA Blood Mini Kit (QIAGEN) and library preparation for RNA-Seq was performed using the Ovation RNA amplification system V2 (NuGEN) and Ultra Low Library System V2 were used (NuGEN). Briefly, 10-500pg of total RNA was first subjected to first strand cDNA synthesis using oligo-dT plus selective priming that targets non-ribosomal RNA sequences in the transcriptome. Nucleotide analog and the original template RNA were degraded, leaving only single stranded antisense cDNA fragments (average size of 230 nucleotides). The fragments are primed using a random octamer with the forward adaptor attached to the 5′ end. Following end repair, the reverse adaptor was ligated to the free end of the now double stranded cDNA, which was enriched for coding and regulatory sequences. A dedicated read barcode design was used for sample identification. Final amplification PCR yielded the strand-specific cDNA libraries. These libraries were sequenced as 100bp paired end reads using an Illumina HiSeq 2000.

DNA was extracted from mucosal biopsies using the AllPrep MiniKit (Qiagen), according to the manufacturer’s instructions and bisulfite—converted using Zymo DNA methylation Gold kit (Zymo Research). Genome-wide DNA methylation was profiled using the Illumina EPIC BeadChip platform (Illumina, Cambridge, UK).

Blood samples were processed as described.12 (link) In brief, PBMC were obtained using density gradient centrifugation. Half underwent sequential positive selection using CD14+ and CD4+ microbeads (Miltenyi) to yield monocytes and CD4+ T cells. The remainder underwent positive selection using CD19+ and CD8+ microbeads (Miltenyi) to yield CD8+ T cells. Neutrophils were isolated from the red cell pellet by lysis followed by CD16+ microbead (Miltenyi) selection. Separation purities were monitored using flow cytometry as previously reported.10 (link) Lysed samples were kept in RLT buffer (Qiagen) at −80°C until required, and then RNA was extracted using the AllPrep Mini kit (Qiagen) and hybridised to Affymetrix HuGene 1.1 microarrays according to the manufacturer's protocols. Singaporean samples were processed in Singapore using the same protocol and then shipped to Cambridge as lysed samples in RLT buffer, with comparable separation purities and RNA quality.

Genomic DNA was extracted from PTC tissues and cell lines using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and the AllPrep Mini Kit (Qiagen) according to the manufacturer protocols, respectively. The mutational hotspots in TERT promoter region and the BRAF V600E mutational status was analyzed as described previously [9 (link),11 (link)]. Sanger sequencing was applied for analyses of the PLEKHS1 promoter mutation and performed at the KIGene core facility, Stockholm, Sweden, using the following primers: 5′-GAA TCC TCG GGA CAA GGC ACT-3′ (Forward) and 5′-GTC AGT CCT ATT TCC CTC TGA CT-3′ (Reverse). PCR was run for 40 cycles with an annealing temperature at 60 °C, which generated PCR products of 241 bp (Figure 1A). Sequencing data were analyzed by visual inspection of chromatograms and manual examination.

Total RNA was extracted from liver samples using a commercially available extraction kit (AllPrep Mini kit, Qiagen, cat 80204) as per the manufacturer’s instructions. Genomic DNA contamination was removed from each sample via treatment with RNase-free DNase (Invitrogen Life Technologies; Auckland, NZ) according to the manufacturer's instructions. RNA quantity and purity were analysed using a NanoDrop spectrophotometer (ND-1000; BioLab Ltd) using NanoDrop software (version 3.1.2). All RNA samples were stored at -80°C until required.
5μg of total RNA was used for first strand cDNA synthesis using a commercially available cDNA synthesis kit (Superscript® VILO™, Invitrogen, cat 11754–250) and a standard thermocycler (GeneAmp® PCR System 9700, Applied Biosystems, California, USA), under the following cycling conditions: an initial denaturation stage of 5 minutes at 96°C, followed by 30 cycles of 30 seconds each of 96°C (denaturation), 60°C (annealing stage) and 72°C (extension stage). cDNA was stored at -20°C.