X cell electroporation system

To obtain stably transfected A549 cells, we used the Sleeping Beauty transposase (SB100) [20 (link)]. The pCMV(CAT)T7-SB100 vector was a gift from Dr. Zsuzsanna Izsvak (Addgene plasmid # 34879) and pT2/HB was a gift from Dr. Perry Hackett (Addgene plasmid # 26557). The coding sequences of CTDSP1, 2, or L were inserted into the pT2/HB plasmid (cloning was conducted by Evrogen, Moscow, Russia). The resulting plasmids (Supplementary Figure S2A), were transfected into A549 cells together with the pSB100 vector encoding the transposase, and pTagRFP vector (FP141, Evrogen, Russia) encoding red fluorescent protein (RFP) using Bio-Rad X-Cell Electroporation System. Next day after transfection, RFP-expressing cells were sorted using S3 cell sorter (Bio-Rad) and cloned by limiting dilution into 96-well plates (Costar, U.S.A.).
Alternatively, protein-coding DNA sequences of the genes CTDSP1, 2, or L were joined with the enhanced green fluorescent protein (EGFP) gene coding sequence through the T2A linker and cloned into the pT2/HB vector (Supplementary Figure S2B). It allowed us to measure the expression of the three proteins by EGFP fluorescence. Then, A549 cells were transfected with the resulting constructs together with pSB100 using Bio-Rad X-Cell Electroporation System.

For NvGcm and NvEaat1 RNA interference by transfection, N. vectensis embryos were electroporated as described in the previous report^{59 (link)} with the following modifications. No Ficoll was added to the brackish water medium because it decreased embryo survival. siRNA was used instead of shRNA due to the simplicity of siRNA manufacturing. Egg masses were collected, de-gelled, and fertilized prior to transfection. Electroporation was carried out using a Gene Pulser Xcell electroporation system (BIO-RAD).
NvGcm-specific and NvEaat1-specific siRNAs sequences were designed to knock down those genes (Supplementary Table 4). Updated N. vectensis genome assembly (NCBI, 20 Mar 2021) distinguishes two Gcm genes: 5,495,825 and 5,504,408 (IDs). Because of the high similarity of the sequences (98%), it is technically difficult to separate the two genes and to analyze their protein functions independently. The two NvGcms are not distinguished in single-cell data^{10 (link)}. In our study we do not separate them either. Instead, NvGcm-targeted siRNAs and NvGcm primers recognize both paralogs. Control embryos were electroporated with a negative control siRNA that was not complementary to any part of the genome (Supplementary Table 4). An siRNA concentration of 500 ng/uL was used. 4dpf N. vectensis planulae were collected for RNA extraction and knock-down efficiency assessment employed RT-qPCR.