Maxtract tube

RNA from breast cancer cell lines was extracted using RNeasy Plus Mini kit (Qiagen) according to manufacturer’s instructions. For breast tissue samples, RNA was extracted by submerging tissue into liquid nitrogen, grinding into a fine powder then using the RNeasy Lipid Tissue Mini Kit (Qiagen) in combination with Maxtract tubes (Qiagen) according to manufacturer’s instructions. RNA was quantified using Nanodrop (Thermo Fisher) and quality was assessed by agarose gel separation. cDNA was then synthesized from 1 µg or 500 ng of total RNA using a Vilo cDNA kit (Invitrogen, via Biosciences, Dublin, Ireland).

Pulverized uterus tissue was incubated for five minutes in trizol reagent. After centrifugation at 10.000×g for 10 min at 4°C, the supernatant was transferred to a Maxtract tube (Qiagen, Copenhagen, Denmark), and chloroform and RNase free water was added. Following centrifugation at 12.000×g for 10 min at room temperature the colorless water phase was transferred to a clean tube and added isopropanol. After 10 min incubation the mix was transferred to a spin column and DNase treated using SV total RNA purification kit (Promega, Nacka, Sweden). The following RNA wash was performed with SV total RNA purification kit (Promega, Nacka, Sweden) according to the guidelines from the manufacturer. Total RNA was eluted in H₂O. Concentration and purity of the RNA was measured by spectrophotometry on a nanodrop and bio-analyzer. RIN values above 7 were accepted for further analysis. The RNA was kept at −80°C until further use.

Genomic DNA (gDNA) was extracted from LD and ST muscle tissue by overnight digestion at 50 °C in digestion buffer [100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% (w/v) SDS, 80 μg/ml RNaseA and 0.1 mg/ml Proteinase K]. The gDNA was then extracted with Phenol:Chloroform:Isoamyl Alcohol (25:24:1 (v/v)) using the Maxtract Tube (Qiagen), precipitated with 0.5 volume of 7.5 M ammonium acetate and 2 volume 100% ethanol, resuspended in nuclease-free H₂O (Ambion) and stored at -20 °C. The gDNA was then quantified using the Quant-iT PicoGreen dsDNA assay kit (Life technologies) according to manufacturer’s instructions and its integrity was evaluated on a 0.8% (w/v) agarose gel.

Directly after cell sorting using FACS, samples were incubated for 30 min at 37°C. Total volume was filled to 250 μl using RNase-free H2O (Thermo Fisher Scientific) followed by the addition of 750 μl Trizol LS (Thermo Fisher Scientific). Samples were mixed by inversion (five times). After a 5 min incubation step at RT, the entire solution was transferred into a MaXtract tube (QIAGEN). A total of 200 μl chloroform (Sigma-Aldrich) was added, followed by three times 5 s vortexing and 2 min incubation at RT. Samples were centrifuged for 2 min at 12 000 rpm at 18°C. The supernatant was transferred to a new tube and isopropanol (Sigma-Aldrich) was added in a 1:1 ratio. For better visibility of the RNA pellet 1 μl GlycoBlue (Thermo Fisher Scientific) was added and the entire solution was mixed by vortexing (3× 5 s). Samples were left for precipitation o/n at −20°C. After precipitation samples were centrifuged for 20 min with 14 000 rpm at 4°C. The supernatant was removed and the RNA pellet was washed with 70% ethanol, followed by a 5 min centrifugation step (14 000 rpm at 4°C). The RNA pellet was resuspended in 12.5 μl RNase-free H₂O. RNA quality was analysed using RNA 6000 Pico kit (Agilent) following the manufacturer’s instructions. The RNA samples were stored at −80°C until further use. RNA sequencing was performed by VBCF GmbH on Illumina platforms.

Upon cell sorting, samples were incubated for 30 min at 37 °C. Total volume was adjusted to 250 µl using RNase-free H₂O (Thermo Fisher Scientific) followed by addition of 750 µl Trizol LS (Thermo Fisher Scientific). Samples were mixed by inverting five times. After a 5-min incubation step at RT, the entire solution was transferred into a MaXtract tube (Qiagen). Two hundred microliters chloroform (Sigma-Aldrich) was added, followed by three times 5-s vortexing and 2-min incubation at RT. Samples were centrifuged for 2 min with 12,000 rpm at 18 °C. Supernatant was transferred to a new tube and isopropanol (Sigma-Aldrich) was added in a 1:1 ratio. For better visibility of the RNA pellet, 1 µl GlycoBlue (Thermo Fisher Scientific) was added and entire solution was mixed by vortexing (3 × 5 s). Samples were left for precipitation o/n at −20 °C. After precipitation samples were centrifuged for 20 min with 14,000 rpm at 4 °C. Supernatant was removed and RNA pellet was washed with 70% ethanol, followed by a 5-min centrifugation step (14,000 rpm at 4 °C). RNA pellet was resuspended in 12.5 µl RNase-free H₂O. RNA quality was analyzed using Bioanalyzer RNA 6000 Pico kit (Agilent) following the manufacturer’s instructions. RNA samples were stored at −80 °C until further use.