Total RNA was extracted using the Norgen single-cell RNA purification kit (Norgen, 51800) with on-column DNase digestion (Norgen, 25710). Four-hundred nanograms of total RNA was transcribed to cDNA using the reverse transcription system (Promega, A3500) and random primers. qPCRs were performed using the SYBR Green PCR master mix (Applied Biosystems, 4309155) with standard cycling conditions. Unless indicated otherwise, data were normalized to Gapdh expression levels.
For splice isoform RT-qPCRs, the specificity of PCR products was analyzed by sequencing of PCR products. PSI values were calculated as described previously (Han et al. 2013 (link); Camacho Londoño and Philipp 2016 (link)) by the following formula:
qPCR primer sequences are inSupplemental Table S11 .
Sequencing library preparation was performed using either ScriptSeq 2 (Epicentre, SSV21106) or TruSeq 2 (Illumina, RS-122-2001) kits.
For splice isoform RT-qPCRs, the specificity of PCR products was analyzed by sequencing of PCR products. PSI values were calculated as described previously (Han et al. 2013 (link); Camacho Londoño and Philipp 2016 (link)) by the following formula:
qPCR primer sequences are in
Sequencing library preparation was performed using either ScriptSeq 2 (Epicentre, SSV21106) or TruSeq 2 (Illumina, RS-122-2001) kits.
