Fig. 6

Derivation of MALAT1 knockout clones. a MALAT1 RNA expression levels of three HCT116 clones lacking the upstream promoter, deleted using the MALAT1_A DECKO construct. KO, homozygous knockout. Expression detected using Primer Set 1 (see below) and values are relative to a control cell clone expressing pDECKO GFP gRNAs. Error bars indicate the standard deviation of three technical replicates. b As for (a) in a heterozygous clone for the major promoter, deleted using the DECKO_C construct. Het, heterozygote. c MALAT1 expression in HEK293T cells heterozygous or homozygous for the major promoter, deleted using the DECKO_C & D constructs. Expression detected using Primer Set 1. d As for (c) in HeLa clones, using the DECKO_C & E constructs. e Semi-quantitative PCR carried out on the same HEK293T clones that in Fig. 6c. “RT-“indicates control samples where Reverse Transcriptase was omitted. f-g qRTPCR using three additional downstream primer pairs (see Fig. 2a), illustrating knockdown of MALAT1 RNA throughout the gene, for the HEK293T and HeLa MALAT1_C clones. h Sequence analysis of mutant junctions. MALAT1_A KO 3 contains one extra C in the deletion point. MALAT1_A 1, MALAT1_A 2 and MALAT1_A 4 have the expected deletion, with no indel. Both gRNA are in antisense direction. Expected cut location is marked with vertical bar. i Proliferation assay comparing the growth rates of control GFP and MALAT1 HEK293T knockout and heterozygous clones. Error bars indicate standard deviation of five cell wells. **, P < 0.01/***, P < 0.001 by Student’s one-sided, paired t test