Rnaseout recombinant ribonuclease inhibitor

Nuclei were isolated from frozen VC tissue. Tissue was manually homogenized using a two-step Dounce homogenizer (A and B) (Sigma #D9063) in NIMT buffer, containing (in mM: 250 sucrose, 25 KCl, 5 MgCl₂, 10 Tris-Cl pH 8, 1 DTT; 1:100 dilution of Triton X100, Protease Inhibitor Cocktail [Sigma #P8340]; and 1:1000 dilution of RNaseOUT Recombinant Ribonuclease Inhibitor [Thermo #10777019]; SUPERase• In RNase Inhibitor [Thermo #AM2694]) on ice. Homogenized samples were mixed with 50% iodixanol (OptiPrep Density Gradient Medium; Sigma #D1556) and loaded onto 25% iodixanol cushion, and centrifuged at 10,000 g for 20 min at 4°C in a swinging bucket rotor (Sorval HS-4). Pellets resuspended in ice-cold DPBS (HyClone) with 1:1000 dilution of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo #10777019); SUPERase• In RNase Inhibitor (Thermo #AM2694). Nuclei were then incubated for 7 min on ice with Hoechst 33342 solution (20 mM) (Thermo #62249) (final concentration 0.5 µM), followed by centrifugation at 1000 g for 10 min at 4°C to pellet nuclei. Pellets were resuspended in blocking buffer containing DPBS with RNAse inhibitors, and 1:10 dilution of pure BSA, and blocked for 30 min on ice. NEUN-Alexa488 pre-conjugated antibody (Millipore #MAB377X) was then added at 1:1000 dilution and incubated for at least 1 hr on ice before proceeding to flow cytometry sorting.

Purification of total RNA from cells was performed using the QIAshredder and RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription reactions were performed using SuperScript II Reverse Transcription and RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific) to obtain cDNA. qPCR analysis was performed using Power UP SYBR Green Master Mix (Thermo Fisher Scientific) and 7500 Fast Real-Time PCR System (Applied Biosystems). The mRNA levels of the genes of interest were normalized using GAPDH mRNA. Normalized mRNA levels were compared between siRNA-treated and control conditions using the comparative Ct method. The following primers were used: GAPDH forward 5′-TGA​TGA​CAT​CAA​GAA​GGT​GGT​G-3′, reverse 5′-TCC​TTG​GAG​GCC​ATG​TGG​GCC​A-3′; ACTR3 forward 5′-CGA​TAT​GCA​GTT​TGG​TTT​GG-3′, reverse 5′-TTT​GGT​GTG​GCA​TAC​TTG​GT-3′; ACTR3B forward 5′-GCC​CGC​TGT​ATA​AGA​ATG​TCG-3′, reverse 5′-AAT​CCC​TGA​ACA​TGG​TGG​AGC-3′; MICAL2 forward 5′-GTG​CAC​GAA​CAC​CAA​GTG​TC-3′, reverse 5′-CCA​GAG​GTG​TAG​CAC​GTT​GT-3′; and coronin 1C forward 5′-GGT​TTC​TCG​TGT​GAC​CTG​GG-3′, reverse 5′-TCA​ATT​CGA​CCA​GTC​TTG​TGC-3′.

Biotin-14-CTP (Thermo Fisher Scientific, Cat# 19519016) labeled RNA was in vitro transcribed using MEGAshortscript™ T7 High Yield Transcription Kit (Thermo Fisher Scientific, Cat# AM1354) according to the manufacturer's instruction. After purification with RNeasy Plus Mini Kit (Qiagen, Cat# 74136), 2 µg biotinylated RNAs was incubated with 20 µL pre-washed Dynabeads™ M-270 Streptavidin (Thermo Fisher Scientific, Cat# 65305) in 1 mL buffer (20 mM Tris–HCl (pH7.5), 100 mM KCl, 5 mM MgCl₂, and 0.5% NP-40), and incubated at 4 °C for 1 h with gentle agitation. 450 µg mIEC cytoplasmic protein lysate was added to the washed RNA-probe coupled dynabeads, and incubated in 1 × TENT buffer (10 mM Tris–HCl (pH8.0), 1 mM EDTA (pH 8.0), 250 mM NaCl, and 0.5% Triton X-100) with SIGMAFAST™ protease inhibitor cocktails and RNaseOUT™ Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific, Cat# 10777019) at 4 °C for 8 h. After washed with 1 × TENT buffer, the proteins interacted with mouse lgr5 probes were eluted using 40 µL SDS buffer and detected by immunoblot assay. Biotinylated Gapdh RNA was used as a pull-down control. All RNA-probes are listed in Table S3.