Analysis of pre-mRNA splicing trans-regulation in human lymphoblastoid cell lines

To understand the role of trans factors in the regulation of splicing and alternative splicing, we knocked-down 22 RNA-binding proteins previously shown or suspected to be involved in the regulation of splicing (“splicing factors”). We performed knockdown experiments in triplicate in the Hapmap lymphoblastoid cell line GM19238 using small interfering RNAs (siRNAs). RNA was extracted 72 hours after knockdown for RNA-seq library preparation. The following splicing factors were analyzed: ACIN1, ADAR, HNRPA2B1, HNRPF, HNRPH1, HNRPH2, HNRPK, HNRPL, HNRPR, PCBP2, PTBP1, RBM23, RBM39, RBMX, RNPS1, SRSF1, SRSF3, SRSF4, SRSF8, SRSF9, SRSF10 and SYNCRIP. Seven controls were generated with non-specific siRNA probes transfection. RNA-seq libraries were multiplexed and sequenced on 3 different flow-cells on an Illumina HiSeq 2000 sequencer (single-end 107 bp long reads). Overall design: 73 samples, including 3 replicates for each of the 22 knockdowns and 7 controls