title: SHAPE-seq data of LINC01958 in glioblastoma cells
design: The in vivo RNA secondary structure was determined by SHAPE-seq using SHAPE reagent, NAI-N3, as described previously, The indicated GBM cells were washed once with PBS and harvested by spinning at 1000 g at 25 C for 2 min. Cell pellets were resuspended in 500 ul of 100 mM NAI-N3 or 500 ul of DMSO and incubated at 37 C for 10 min. Cells were then immediately centrifuged at 2500 g at 4 C for 1 min and removed the supernatant to stop the reaction. Cell pellets were resuspended in 500 ul of PBS and then added into 6 ml of Trizol LS. After homogenize by vortex, 1.2 ml of chloroform was added and vortex again. RNA was purified using an Hipure Total RNA plus Micro Kit according to the manufacturer's protocol. Reverse transcription of 12 ug of RNA was performed using the LINC01956-specific RT primers according to the manufacturer's protocol of Induro Reverse Transcriptase (New England). Then, 2 pl of 2.5 M NaOH was added in samples and incubated at 95 C for 1 min to stop the reaction. The sample solution was neutralized by adding 1 ul of 5 M HCI. Next, the cDNA was purified with a MinElute Reaction Cleanup Kit (QIAGEN). LINC01956 was amplified with KOD-plus-neo (Toyobo) using LINC01956-specific primers. PCR products were purified using Agencourt AMPure XP beads (Beckman). The purified DNA samples were subjected to PacBio library construction by Novogene using a PacBio Sequel 3.2.