GSM6432846: SELEX PTBP1 rep2; synthetic construct; OTHER
The input ssDNA library for SELEX was synthesized. The library was amplified by PCR (5 cycles) to generate templates for the initial in vitro transcription of the ssRNA library. To pull down PTBP1 and its interacting RNA, 40 µl of protein A/G magnetic beads coupled with anti-PTBP1 antibody were added to the ssRNA pool and then rotated at room temperature for 1 h. To elute the bound RNA, the beads were washed three times with 1 ml of binding buffer and then resuspended in 50 µl of TE buffer, 50 µl of 2 × Proteinase K buffer (0.02 M Tris-HCl, pH7.8, 0.02 M EDTA, 1% SDS), and 1 µl of Proteinase K and incubated at 50 °C for 30 min. RNA was extracted with Trizol LS, reverse transcribed into cDNA, and amplified by PCR. The PCR products were fractionated on the 2% agarose gel and purified with Gel Extraction Kit (Qiagen, 28604). This whole process was performed three times. In the third round, cDNA was amplified with index-containing primers compatible with Illumina HiSeq X Ten.