RNA-seq of Homo sapiens: Diabetic Foot Ulcer
Frozen samples in RNAlater were thawed on ice. The tissue was removed and placed inside an empty, sterile Petri plate. A scalpel was used to cut a piece of tissue with a volume of approximately 0.5 cm3. RNA extraction was then performed chloroform/phenol phase separation. The tissue was placed in a 2 mL microtube (Sarstedt, Germany, Cat: 72.693.005) filled one-third with 2mm and 0.1 mm diameter zirconia beads (Biospec, USA). One milliliter of RNABee (Amsbio, UK, cat: CS-501) containing 10 uL/mL -mercaptoethanol was added to the tubes. The tubes were placed in a MagNA Lyzer instrument (Roche) and homogenized at maximum power for 3 x 30 seconds. Tubes were placed on ice for 1 min after each homogenization step. The tubes were shaken vigorously for 30 sec, incubated for 5 min on ice, and then centrifuged at 13,000 x g for 30 min at 4 C. The upper aqueous phase was collected and placed in a new, 1.5 uL DNA Lo-Bind centrifuge tubes (Eppendorf AG, Germany, cat: 0030108051). Ice-cold ethanol (0.5mL) and 2 l of 5 mg/mL linear acrylamide was added. The tubes were inverted several times and stored at -80 C overnight. The samples were then thawed on ice and centrifuged (13,000 x g, 30 min, +4 C). The supernatant was discarded and the pellet was washed twice with fresh, ice-cold 75 % ethanol. The pellet was then resuspended in 20  65 uL nuclease free water. RNA concentration was quantified with a NanoDrop spectrophotometer. Contaminating DNA was removed by combining ~2.5 ug RNA with 3 uL RQ-1 RNAse Free DNase (Promega, USA), 3uL DNAse buffer solution, 1uL RiboGuard RNAse inhibitor (Lucigen, USA, cat: RG90925), and nuclease free water to a final volume of 30uL. The DNAse-treated RNA was then re-purified with the RNABee protocol described above and then stored at 80C. Ribosomal RNA depletion was performed either with the 10:1 human:pan-prokaryote riboPOOLs (siTOOLS Biotech, Germany, protocol version: 1.4.2) for the P*** samples or the RiboZero Gold Epidemiology rRNA depletion kit (Illumina, USA), for the KK_** samples. The NEBNext Ultra II RNA library preparation kit (New England Biolabs (NEB), USA, cat: E7775S) was used to prepare cDNA libraries for the P*** samples. The NEBNext Ultra II non-directional RNA library preparation kit (New England Biolabs, USA, cat: E7775S) was used to prepare cDNA libraries for the P*** samples. The NEBNext Small RNA Library Prep was used to prepare the KK_** samples. Concentrations and quality of the final libraries were assessed with a Qubit 4 fluorometer and Agilent Bioanalyzer. Samples were sequenced either on an Illumina HiSeq4000 or Novaseq sequencing instrument.