Deparaffinization buffer

LCM was performed as previously described (Ramirez et al., 2015 (link)). Briefly, formalin-fixed paraffin embedded tissue blocks from DFU, were sliced into 16 to 20 sections (8-10μm thick), collected on Arcturus PEN-membrane glass slides (Life Technologies, Carlsbad, CA) and dried at 37°C for 1-2 hours. LCM was carried out on an Arcturus Veritas laser capture microdissection instrument and the epidermis was captured on CapSure® Macro LCM Caps (Life Technologies). Caps were then transferred into a tube containing 60μl of deparaffinization buffer (QIAGEN Inc., Valencia, CA) and total RNA, including the miR fraction, was extracted using the FFPE miRNeasy kit (QIAGEN Inc.) according to the manufacturer’s instructions.
The RNA concentration of the samples was measured using a NanoDrop 2000 (NanoDrop products, Wilmington, DE). MiR profiles of epidermis of 6 DFUs were generated using the miR Ready-to-Use PCR panels V2 (Exiqon) following the manufacturer’s specifications. miR-15b-5p expression was calculated using the ΔΔCT method with SNORD49 as reference, and comparing the CTs of DFUs and previously published FS (NFS and DFS) array data (Ramirez et al., 2015 (link)).

For the frozen normal, Barrett’s esophagus and EAC samples, DNA was extracted from the tissue using either the DNeasy kit (Qiagen) or the AllPrep DNA/RNA Mini Kit (Qiagen), according to the manufacturer’s instructions. DNA was extracted from blood samples using the Nucleon^™ Genomic Extraction kit (Gen-Probe,) according to the manufacturer’s instructions. For the Barrett’s esophagus FFPE biopsy samples, genomic DNA was extracted from the rest of the diagnostic biopsy and each diagnostic biopsy was extracted separately. For the Cytosponge sample, genomic DNA was extracted from 8 × 10 μm sections of the processed Cytosponge FFPE clot. The FFPE samples were extracted using Deparaffinization Buffer (Qiagen) and the QIAamp FFPE DNA Tissue Kit (Qiagen). The protocol was followed as described by the manufacturer with the exception that samples were incubated at 56°C for 24 hours instead of the described 1 hour, and 10 μl of extra Proteinase K was added to the samples roughly half way through the 24 hour incubation. For all sample types the DNA was quantified using the Qubit^™ dsDNA BR or HS Assay Kits (Life Technologies).

FFPE tumor blocks were sectioned, slide-mounted, and H&E-stained to mark tumor boundaries. Adjacent normal tissue was removed by scalpel. Isolated tumor sections were placed in Qiagen deparaffinization buffer at 56°C for 5 minutes. Samples were incubated in Proteinase K and crosslink reversal buffer (Qiagen Buffer FTB) for 1 hour at 56°C for protein digestion, followed by 90°C for 1 hour to reverse-crosslink DNA. 35 ul of uracil-N-glycosylase (UNG) included with the kit was added to each sample and incubated at 50°C for 1 hour to correct fixation-induced cytosine deamination. Samples were treated with RNAse A, followed by complete lysis (Qiagen Buffer AL & ethanol). DNA was captured, washed, and eluted on Qiagen gDNA capture columns. DNA was subsequently treated with NEBNext FFPE Repair Mix and purified by Ampure XP beads. Agilent Tapestation was used to determine DNA size and DNA was submitted for whole-exome sequencing. All FFPE tumor samples were paired with patient-matched normal DNA. 25 normal samples were pre-HSCT peripheral blood, 10 were primary fibroblasts, and 6 were normal tissue FFPE samples. Novogene performed exome capture (Agilent SureSelect XT V6 & IDT xGen Exome) and library preparation. FFPE tumor samples were 2×150bp sequenced to 12Gb of raw output, and normal samples were sequenced to 6Gb of raw output.