So142

Whole RNA was extracted using the Monarch total RNA Miniprep kit and associated protocol (NEB, T2010). Briefly, cells were lysed and run through spin columns to remove genomic DNA and to purify RNA. RNA was eluted from columns using nuclease free H₂O (Severn Biotech Ltd, 20‐9104‐05). Concentration of RNA was quantified on nanodrop (Thermofisher). cDNA was synthesized using 1 μg whole RNA, 1 μl of 10 mM deoxyribose nucleotide triphosphates (DNTPs) (Thermofisher, R0192) and 1 μl of 20X random hexamer primers (ThermoFisher, SO142). Samples were made up to 13 μl with nuclease free water. RNA, DNTPs and random hexamer primers were incubated at 65°C for 5 min, before being placed on ice. After ~1 min, 2 μl of 100 mM 1,4‐dithiothreitol (DTT) and 4 μl 5X first strand buffer (250 mM tris(hydroxymethyl)aminomethane (Tris)‐HCl (pH 8.3), 375 mM KCl, 15 mM MgCl₂) were added per sample. Samples were left at room temperature for ~3 min before adding 1 μl (200 units) of Superscript II Transcriptase (Invitrogen, 18064022) was then added per sample. Total volume of sample was 20 μl and these were placed in a thermocycler (Biorad) and cDNA generated using the following conditions: 25°C for 10 min, 42°C for 50 min, 72°C for 15 min, 4°C hold.

RNA isolation was performed as previously described according to MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines [17 (link), 30 (link)]. A total of 500 µl peqGOLD TriFast^TM (PEQLAB Biotechnology GmbH, Erlangen, Germany) was added per well and further processed according to the manufacturer’s instructions. The resulting RNA pellet was resuspended in 20 µl nuclease-free doubly distilled water (H₂O_dd; T143, Carl Roth, Karlsruhe, Germany). RNA was quantified using a NanoPhotometer (N60; Implen, Munich, Germany). A total of 100 ng RNA per sample was transcribed into cDNA using 1 µl oligo-dT18 primer (SO131, Thermo Fisher Scientific Inc., Waltham, MA, USA), 1 µl random hexamer primer (SO142, Thermo Fisher Scientific Inc.), 1 µl dNTP mix (L785.2, Roti®-Mix PCR3, Carl Roth), 1 µl RNase inhibitor (EO0381, Thermo Fisher Scientific Inc.), 1 µl MLV-reverse transcriptase (M1705, Promega, Fitchburg, WI, USA), 4 µl 5 × M-MLV-buffer (M1705, Promega) in a total volume of 20 µl by addition of nuclease-free H₂O_dd (T143, Carl Roth). Samples were incubated for 60 min at 37 °C and 2 min at 95 °C. Reverse transcription was performed for all samples at the same time to minimize experimental variation.

Total RNA was extracted by RNeasy Mini Kit (QIAGEN, Hilden, Germany). The amount of the total RNA was measured by microvolume spectroscopy (IMPLEN, USANanoPhotometer^® N60/N50). For the RT-PCR, 5 ug of total RNA for the cDNA synthesis, random hexamer primer (Thermo Fisher, SO142, Waltham, MA, USA), and reverse transcriptase (Thermo Fisher, Waltham, MA, USA) were used according to the manufacturer’s instructions. PCRs were performed by using the SYBR Green method in an ABI 7000 sequence detection system (Applied Biosystems). The primer sequences are listed in Supplementary Materials Table S1.

For cDNA synthesis we mixed 1 μg of RNA with nuclease-free water to get a volume of 11 μl. This compound was applied to a mixture of 4 μl 5xM-MLV-buffer (M1705, Promega, Madison, WI, USA), 1 μl Oligo_dt primer (SO131, Thermo Fisher Scientific, Waltham, MA, USA), 1 μl random hexamer primer (SO142, Thermo Fisher Scientific, Waltham, MA, USA), 1 μl 10 mM dNTP (L785.2, Carl Roth, Karlsruhe, Germany), 1 μl (40 U) RNase Inhibitor (EO0381, Thermo Fisher Scientific, Waltham, MA, USA) and 1μl (200 U) M-MLV Reverse Transcriptase (M1705, Promega, Madison, WI, USA) [6 (link)]. All samples were incubated at 37°C for 1 h and at 95°C for 2 min to inactivate the transcriptase. They were stored at -20°C until use. To minimize experimental variations, all components were prepared as a master mix and cDNA synthesis was performed at the same for all samples.

Total RNA was extracted with QIAzol (79309, QIAGEN) according to the manufacturer’s instructions. Contaminating genomic DNA was removed by treatment with DNAse I (EN0523, Thermo Fisher Scientific) in the presence of Ribonuclease Inhibitor (EO0381, Thermo Fisher Scientific). For cDNA synthesis, 1 μg total RNA from mouse keratinocytes was reverse-transcribed using random hexamer primers (SO142, Thermo Fisher Scientific) and Revert Aid reverse transcriptase (EP0441, Thermo Fisher Scientific). For reverse transcription of RNA from tissue, cDNA was synthesized from 4 μg total RNA using oligo-dT primer (SO132, Thermo Fisher Scientific) and Revert Aid reverse transcriptase. cDNA reaction was performed for 1 hour at 42°C. Relative gene expression was quantified by real-time PCR using the Green master mix from Genaxxon (M3023) and self-designed primers (Supplemental Table 1). Real-time PCR analysis was performed on a Light Cycler 480 II system (Roche) using the following PCR conditions: initial denaturation 15 minutes at 95°C, followed by 40 cycles of 95°C for 15 seconds and 60°C for 45 seconds. Relative mRNA levels were calculated by normalization to the reference gene Actin using the 2^-ΔΔCt method.

For reverse transcription, equal concentrations of RNA were diluted with nuclease-free water (T143.5, Carl Roth, Karlsruhe, Germany) to a volume of 5.5 µL. To each sample, 2 µL of M-MLV reverse transcriptase buffer (M1705, Promega, Madison, WI, USA), 0.5 µL of OligodT (SO132, Thermo Fisher Scientific, Waltham, MA, USA), 0.5 µL of random hexamer primer (SO142, Thermo Fisher Scientific, Waltham, MA, USA), 0.5 µL of dNTPs (L785. 2, Carl Roth, Karlsruhe, Germany), 0.5 µL RNAse inhibitor (EO0382, Thermo Fisher Scientific, Waltham, MA, USA), and 0.5 µL reverse transcriptase (M1705, Promega, Madison, WI, USA) were added. To avoid errors due to pipetting, the mixture was prepared as a master mix for the entire experimental approach, so 4.5 µL of the master mix was added to 5.5 µL of the adjusted RNA sample. After mixing, samples were incubated at 37 °C for 1 h, followed by inactivation at 95 °C heat for 2 min and stored at 4 °C.