Ambion pure link kit

For RNA quantification, cells were lysed in RNA lysis buffer, and total RNA was purified with Ambion Pure-Link kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA). The cDNA template was reverse transcribed from 1000 ng of total RNA with a Cloned AMV First-Strand cDNA synthesis kit (Invitrogen, ThermoFisher Scientific). Quantitative RT-PCR (qRT-PCR) was performed as previously described (37 (link)) with Power SYBR green PCR mix (Applied Biosystems, Thermo Fisher Scientific) and 4 ng cDNA template. qRT-PCR primers (listed in Table 1) were used at a final concentration of 200 nM and designed to span either an intronic-exonic region to detect levels of nascent transcript before splicing to mRNA [heteronuclear RNA (hnRNA)] or to span an exonic-exonic region to detect mature transcript (mRNA). The expression of each target gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as determined in a separate real-time PCR; relative hnRNA and mRNA levels were quantified with the 2^–∆∆CT.

For RNA quantification cells were lysed in RNA lysis buffer, and total RNA was purified using Ambion Pure-Link kit (Invitrogen, ThermoFisher Scientific). The cDNA template was reverse-transcribed from 1000 ng of total RNA using Cloned AMV First-Strand cDNA synthesis kit (Invitrogen, ThermoFisher Scientific). RTqPCR was performed as previously described (Park et al., 2013) using Power SYBR green PCR mix (Applied Biosystems, ThermoFisher Scientific) and 4 ng cDNA template. RTqPCR primers (listed in Supplementary Table 1) were used at a final concentration of 200 nM and designed to span an exonic-exonic region to detect mature transcript (mRNA). Each sample was analised in duplicate and GAPDH was used as a house-keeping gene.

Quantification of mRNA and protein levels was performed by RT-qPCR and western blotting respectively, as described previously^{56 (link)}. Total RNA, extracted using Ambion Pure-Link kits (Thermo Fisher) and was reverse transcribed using QuantiTect RT kit (Qiagen) and random primers. Quantitative PCR was performed using Roche SYBR Green using a Qiagen Roto-Gene Q PCR machine (20′@95 °C; 20′@62 °C; 20′@72 °C). Primers sequences are described in Supplement Table 1. Data were normalised to total RNA. Western blots were performed using a Mini-Protean II system. Proteins were transferred to PVDF membrane using a semi-dry Turbo blotter (Bio-Rad) and detected using ECL and a digital ChemiDoc imaging system (Bio-Rad).