Rneasy micro kit

Serial 8μm cryosections (Leica CM3050S, UK) of OCT-embedded keloid and NS samples were cut onto specialised polyethylene naphthalate (PEN) membrane slides (Carl Zeiss, UK). To differentiate epidermis from dermis, whilst preserving tissue RNA integrity, a rapid staining protocol was performed (LCM Staining Kit, Ambion, Austin TX, USA) according to the manufacturer’s instructions [16 (link), 17 (link)]. Using a P.A.L.M. LCM microscope (Carl Zeiss MicroBeam 4.2, Germany) epidermis and dermis of each sample was laser cut and catapulted away from the slide into separate overhanging microtube caps (AdhesiveCap 200 Opaque, Carl Zeiss Microscopy Ltd, Cambridge, UK). Multiple ‘elements’ were captured from each tissue section of least three sequential sections from each patient, ensuring adequate biological representation. The captured tissue was mixed with lysis buffer (Buffer RLT with 1% 2-mercaptoethanol, RNeasy Micro Kit, Qiagen, UK) and stored at -80°C until extraction according to manufacturer’s instructions (RNeasy Micro Kit, Qiagen, UK). Following extraction, the samples were again stored at -80°C [18 (link)].

Gene expression was analyzed in HPCs (12 UCB and 10 AB; 2,000–70,000 cells), CECs (2 UCB and 1 AB; 600–2,000 cells), control leukocytes (3 UCB and 6 AB, CD34^-CD133^-KDR^-CD45⁺), OECs (3 UCB and 1 AB; 500,000 cells), and HUVECs (2 separate cell lines, 500,000 cells). Cells were lysed in RLT buffer (Qiagen RNeasy micro kit) containing 1% β-mercaptoethanol, vortexed for 1’ and stored at -80°C. RNA was isolated using the RNeasy micro kit (Qiagen). cDNA was synthesized using the qScript cDNA SuperMix kit from Quanta. Due to low numbers of sorted HPCs and CECs, the PreAmp cDNA amplification kit (Quanta) was used (up to 100 genes). Amplified cDNA was diluted 20-fold. RT-PCR was performed following manufacturer instructions (Quanta) and 200nM primers. Pre-amplification was extensively validated to determine whether the correct proportion of transcripts was retained. PCR primers are listed in (S1 Table). In order to analyze the cells at functional level, RT-PCR to 10 angiogenic factors and receptors (apelin, PDFDβ, PDGFR, SCF, FGF, EGF, EGFR, VEGFA, Tie-1 and Tie-2) was carried out for all EPC subsets.

Total RNAs from infected GM-CSF-BMDC were extracted using RNeasy Micro Kit (Qiagen) following manufacturer’s instructions. CLN were stabilized in RNAlater (Qiagen) immediately after sampling. Organs were homogenized in RLT buffer (Qiagen) and then extracted using RNeasy Micro Kit (Qiagen) following manufacturer’s instructions. cDNAs were obtained by using Quantitech Reverse Transcription Kit (Qiagen) following manufacturer’s instructions using 300 ng of RNA as a matrix. qPCR were conducted using a 7500 Fast-Real-time PCR (Applied Biosystem) with SYBER Green (Takara) following manufacturer’s instructions. HPRT was used as housekeeping gene to determine ΔCt. Fold change compared to base line expression in uninfected cells, or control mice was determined using 2^-ΔΔCt method where ΔΔCt = (Ct_target-Ct_HPRT)_infected-(Ct_target-Ct_HPRT)_non-infected as previously described (Papadopoulos et al., 2016 (link)). mRNAs whose expression level was twice as high compared to control were considered as significantly up-regulated. Primers used in this study are listed in Table 1.

We performed LMD using a PALM MicroBeam (Carl Zeiss, Oberkochen, Germany) to collect RNA from the tissue. Briefly, the manufacturer’s recommended slides and collection tubes (AdhesiveCap 500 opaque, Carl Zeiss) were set up, and the samples were collected from the wound margins by carefully cutting the tissue while observing with a 20× magnification objective lens. The tube cap was filled with Buffer RLT (RNeasy microkit, Qiagen, Hilden, Germany) and filled with β-mercaptoethanol to allow the separation of intact RNA using the RNeasy microkit (Qiagen). Total RNA was extracted from the cells or skin tissues according to the manufacturer’s instructions, and then placed in a T100^TM thermal cycler (Bio-Rad, Hercules, CA, USA) along with Maxima™ H Minus cDNA Synthesis Master Mix (ThermoFisher Scientific, Waltham, MA, USA) at 25 °C for 5 min, 55 °C for 10 min, and 80 °C for 10 min to thermally inactivate revertase and produce cDNA.

Total nuclear RNA was extracted from ∼10,000 non-fixed nuclei of each sample using the RNeasy Micro Kit (Qiagen) following the manufacturers instructions. Contaminating genomic DNA was removed from the RNA samples by treatment with DNase I (0.05 U per μl) for 30 min at 37 °C (Thermo Scientific) before the RNA was purified a second time using the RNeasy Micro Kit (Qiagen). The concentration of the nuclear RNA was determined using the RNA 6,000 Pico Kit (Agilent). The double-stranded cDNA was synthesized from ∼500 pg RNA by linear amplification using the SMARTer Ultra Low Input RNA for Illumina Sequencing-HV kit (Clontech) according to the manufacturer's instructions. The concentration and yield of the amplified cDNA was determined using the High Sensitivity DNA Kit (Agilent).