In vitro MS-MaP-seq experiment using RNA purified from differentiating keratinocytes to test the effect of adding DDX50/STAU1/glucose on RNA structure.
Primary human keratinocytes were isolated from fresh surgically discarded newborn foreskin, and progenitor cells cultured in complete Keratinocyte-SFM (Life Technologies, #17005-142) and Medium 154 (Life Technologies #M-154-500). Cells were induced to differentiate by the addition of 1.2 mM calcium for 4 days at full confluence. Total RNA was extracted from day 4 differentiated keratinocytes using RNeasy Plus (QIAGEN, 74136). 1 µg of total RNA was used for each experiment. RNA was diluted to 200ng/µL with RNase free water and heated for 1 min at 95 °C, then placed on ice. RNA was subsequentially diluted in refolding buffer to 90 µL before protein or glucose were added to the mix. A total of 6 conditions were explored (DMS was not added at this step): 1. No DMS+200ng BSA; 2. DMS+200ng BSA; 3. DMS+200ng BSA+1 mM glucose; 4. DMS+200ng DDX50; 5. DMS+150ng STAU1; 6. DMS+200ng DDX50+150ng STAU1+1mM glucose. Mixtures were incubated at 37 °C for 10 min on thermomixer. After incubation, 10 µL 1.7M DMS (diluted in ethanol) or 10 µL ethanol (for no DMS samples) was added to the mixtures and RNA labeled by incubation at 37 °C for 5 min on a thermomixer while shaking at 500rpm. To quench, 60 µL ß-mercaptoethanol was added to the mixture immediately and labeled RNA was purified. DMS labeled RNA was first purified using the Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen, 61012). 12 µL poly(A) beads per sample were washed twice with binding buffer (100 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM EDTA, 1% SDS), then added to purified RNA in 600 µL of binding buffer, incubated for 3-5 minutes at room temperature with mixing to bind RNA to the beads, washed with the kit's buffers according to the kit's instructions, and eluted in 11 µL of 10 mM Tris-HCl (pH 7.5) by heating at 65-80°C for 2 minutes. Purified poly(A) RNA was converted to a sequencing library with the SMARTer® Stranded Total RNA-SeqKit v3 Pico Input Mammalian kit (Takara, 634485) following the manufacturer's instructions and using a 1.5 min 94°C initial incubation in First-Strand buffer and 7 cycles during the first PCR reaction.
NIAMS-phs003766
a_Glc_dms10