GSM8257631: WT_siInts8_unt_1; Homo sapiens; OTHER
RNA was extracted by TRIzol (Thermo Fisher Scientific, 15596026) accordingly to the manufacturers instructions. As a control for equal sample preparation, we spiked-in S. cerevisiae (strain BY4741, MATa, his3D1, leu2D0, met15D0, ura3D0) 4-thiouracil (4TU)-labelled RNA. For purification of 4sU labelled RNA, 100 ug mammalian 4SU labelled RNA was spiked-in with 1ug of 4tU-labelled S. cerevisiae RNA. The 101 ug RNA was fragmented by addition of 20 uL of 1 M NaOH to fragment RNA and left on ice for 20 min. Fragmentation was stopped by addition of 80 uL 1 M Tris pH 6.8 and cleaned up twice with Micro Bio-Spin‚P-30 Gel Columns (BioRad, 7326223). Biotinylation of 4sU-residues were carried out in a total volume of 250 ul, containing 10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 50uL DMF and 5mg MTSEA biotin-XX linker (Biotium, BT90066) for 30 min at room temperature in the dark. RNA was then purified by phenol:chloroform extraction, denatured by 10 min incubation at 65C and added to 200 uL uMACS Streptavidin MicroBeads (Miltenyi, 130-074-101). RNA was incubated with beads for 15 min at room temperature and beads applied to a uColumn in the magnetic field of a uMACS magnetic separator. Beads were washed twice with pull-out wash buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween20). 4sU-RNA was eluted twice by addition of 100 mM DTT and RNA cleaned up using the RNeasy MinElute kit (Qiagen, 74204). Libraries for RNA sequencing were prepared using the KAPA RNA HyperPrep Kit (KR1350) with modifications. 75 ng of RNA per sample were mixed with FPE Buffer, but fragmentation procedure was omitted and RNA was instead denatured at 65C for 5 min. The rest of the procedure was performed as recommended by the manufacturer, with the exception of SPRI bead purifications: after adaptor ligation, 0.95x and 1x SPRI bead-to-sample volume ratios were used (instead of two rounds of SPRI purification with 0.63x volume ratios). This was done to retain smaller (150-300 bp) cDNA fragments in the library which would otherwise be lost in size selection. The libraries were quality controlled by electrophoresis on a Tapestation system (Agilent), quantified by Qubit (Thermofisher).