title: RNA-seq of Lingula anatina late gastrula - treated with low concentration LDN BMP inhibitor 3
design: BMP signalling manipulation experiments were deployed to reveal the function of the BMP pathway during L. anatina development. Two separate small molecule inhibitors were used to block the BMP pathway: LDN193189 (LDN; Stemgent 04-0074) and K02288 (K02; Tocris 4986).Exogenous mouse recombinant BMP4 (mBMP4; R&D Systems 5020-BP) was used to over-activate the BMP pathway. Manipulations were applied for two different durations, from the early blastula stage (5 h post fertilisation, hpf) to either the late gastrula (10 hpf) or the 1-pair-cirri larval stage (2427 hpf). Two doses (high and low) of each manipulator were applied to the late gastrula experiments: LDN (4 and 8 M), K02 (250 and 500 nM) and mBMP4 (100 and 200 ng/mL). One dose was applied to the 1-pair-cirri larva experiments: LDN (2 M), K02 (250 nM) and mBMP4 (200 ng/mL). Controls were run for each manipulator with the vehicle only (bovine serum albumin (BSA) or dimethyl sulfoxide). RNA was extracted for three biological replicates of each condition using TRIzol and sequenced using the Illumina HiSeq 4000 platform.