Rneasy mini column

RNA was modified and collected from mESCs using the same conditions as previously published for mammalian cell modification (Smola et al. 2015 (link)). Old media was removed and cells were washed once with PBS. Of note, 900 µL of fresh medium was replaced on the cells. One hundred microliters of 10× SHAPE Chemical in DMSO, or pure DMSO, was then added to each 900 µL sample at the following final concentrations: 100 mM FAI, 100 mM NAI, 100 mM NAI-N₃, 10 mM 1M7, and 100 mM 1M7. Cells were modified for 5 min at 37°C. Media were removed, and RNA was purified by TRIzol and chloroform preparation followed by RNeasy Mini Column (Qiagen).
E. coli cells were modified with SHAPE reagent using the conditions as written in McGinnis et al. (2015) (link). Cells in LB medium were modified for 5 min at 37°C with SHAPE reagent dissolved in anhydrous DMSO. Final concentrations of SHAPE reagents were as follows: FAI, NAI, NAI-N₃ at 30 mM and 1M7 at 5 mM, all with a final DMSO concentration of 3% (v/v) in LB. Modified RNA was extracted from bacteria by lysis as described earlier, followed by TRIzol chloroform preparation and RNeasy Mini Column (Qiagen).

For fresh tissue collection, animals were terminally anesthetized with CO₂ 3 days after CFA or sham surgery. The spinal cord segment corresponding to the lumbar area was rapidly removed, and the ipsilateral dorsal horn quadrants L4 to L6 were dissected out and frozen on dry ice. Samples were then stored at −80°C until further processing. Total RNA was extracted using an acid phenol extraction method (TRIzol reagent, RNeasy mini-columns; Quiagen, United Kingdom). RNA concentration was measured using the Nanodrop (Labtech International, Ringmer, United Kingdom).

Human heart samples were purchased from AnaBios Corporation (San Diego, CA, USA), which provides heart organs through the organ procurement organization of the USA in compliance with the Health Insurance Portability and Accountability Act. All personal information of donors is protected. In the current study, three normal hearts were separated into LA, RA, LV, RV, and SAN. These samples were frozen in RNAlater (Thermo Fisher Science, Waltham, MA, USA) at −20 °C. RNAs were extracted in our laboratory using QIAGEN RNeasy mini columns (QIAGEN).

Total RNA was prepared from glomeruli and cell lysates using Qiagen RNeasy mini columns (Qiagen, CA) and then reverse transcribed with Superscript-II reverse transcriptase (Invitrogen, CA). cDNA amplification was performed using SYBR-Green PCR Master Mix (Applied Biosystems) and gene-specific exon–exon junction spanning primers (sequences available upon request) in an ABI-Prism 7900HYT Sequence Detection System and evaluated using S.D.S 2.0 software (Applied Biosystems, CA). Normalization to beta-actin gene.