Nucleobond bac100 kit

BAC clones covering NANOG region of interest were prepared as previously described [45 (link)]. BAC clones were purified with NucleoBond^® BAC 100 kit (Macherey–Nagel 740579). An additional BAC clone was prepared covering the ERCC3 locus used as a control for qPCR normalization.

These mutants were constructed from an HCMV strain Merlin BAC (pAL1111 [63 (link)]) by using the recombineering techniques described previously [63 (link)–66 (link)]. Briefly, a selectable KanR/RpsL/lacZ cassette flanked by appropriate HCMV sequences was transfected into Escherichia coli SW102 cells containing the parental BAC. Clones in which the cassette had recombined homologously into the BAC were selected positively using kanamycin. The inserted cassette was then replaced by the original sequence containing the desired mutation, and clones were selected negatively using streptomycin. BAC DNA was extracted using a Nucleobond BAC 100 kit (Macherey-Nagel) according to the manufacturer’s instructions, and virus was reconstituted by transfection into human fibroblast cells using an Amaxa Basic Nucleofector kit for Primary Mammlian Fibroblasts (Lonza). The complete genome sequences of the viruses were determined by Illumina sequencing in order to ensure that the intended mutations were present and no others. Nucleotides 94297–94302 and 96114–99118 were absent from the ΔTATA and the Δ3’ mutants, respectively (coordinates from GenBank accession no. AY446894.2).

TB40-BAC4-DNA was purified from 400 ml of E. coli overnight culture using the Nucleobond BAC 100 kit (Machery-Nagel) for low-copy plasmid purification. All steps were done according to the manufacturer. Purified BAC-DNA was eluted in 50 μl nuclease-free deionized water, quantified using Nanodrop and stored at 4 °C and under no conditions frozen to avoid DNA fragmentation. Purified BAC-DNA was used to establish long range PCR assays.

We used the phiC31-integration method to generate transgenic flies (Bischof et al., 2007 (link)). All P[acman]-based constructs were integrated into the attP40 landing site to ensure identical genetic backgrounds. As previously described (Stephan et al., 2015 (link)), we transferred final P[acman] constructs to Escherichia coli EPI300 cells and induced high-copy amplification to prepare DNA for injection (Venken et al., 2006 (link), 2009 (link)). Using the NucleoBond BAC 100 kit (Machery-Nagel) DNA was prepared following the manufacturer’s instructions and resuspended in 10 mM Tris (pH 8.0) buffer. Using restriction enzymes DNA fingerprints were generated and analyzed to control DNA integrity. Before injection, DNA was diluted in 10 mM Tris (pH 8.0) to a final concentration of 80 ng/μl.

ESRP1 and ESRP2 antibody specificity was validated in control cell lines with ectopic ESRP1 and ESRP2 protein overexpression (Supplementary Fig. 2). To produce these cells, cDNAs encoding ESRP1/RBM35A (# HG13708-UT, Sino Biological Inc., Wayne PA, USA) and ESRP2 (#HG23639-UT, Sino Biological Inc., Wayne PA, USA) were transformed in competent Escherichia coli cells (One ShotTM Top10, TermoFisher Scientifc, Germany), the plasmid DNA was isolated (NucleoBond BAC 100 Kit, #740579, Macherey-Nagel, Düren, Germany) and transfected to cultivated HeLa cells (15 μg/70% confluence/1500 mm dish) using JetPEI DNA Transfection Reagent (Polyplus-transfection, #101-10 N S.A., Illkirch, France). Transfected cells were harvested after 24 h, centrifuged at 1000×g for 5 min, stabilized in agarose, fixed in 4% buffered formalin overnight and embedded in paraffin (FFPE fixation). Non-transfected HeLa cells were cultivated (37 °C and 5% CO₂) in Dulbecco’s Modified Eagles Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S), harvested and FFPE fixed and served as negative control. For immunohistochemistry, freshly cut section of negative and positive control cell lines were stained as described above.

V. planifolia, H. longidenticulata, G. fumata, P. armeniacum, P. niveum, C. formosanum, M. picturata, and E. pusilla leaves were used as the material for DNA extraction via the cetyltrimethyl ammonium bromide method55 (link). The BAC plasmids for Illumina sequencing were isolated using the NucleoBond BAC 100 Kit (NucleoSpin Blood, Macherey-Nagel, Düren, Germany).

The human TNF BAC clone (RP11-184F16; obtained from Life Technologies, UK) was purified using a Machery-Nagel Nucleobond BAC100 kit and used to transform electro-competent SW102 E. coli (a kind gift from Neal Copeland; Houston Methodist Research Institute, Houston, TX, USA). SW102-TNF positive clones were confirmed by pulsed-field gel electrophoresis analysis. Recombineering was performed using seamLess GalK mediated selection/counter selection strategy, as described previously^{26 (link),48 (link)}. In brief, two targeting cassettes designed to excise the first coding exon of the TNF gene were generated to first incorporate the GalK gene within the BAC, positive recombinants derived by selection on minimal media containing galactose, followed by the removal of GalK and integration of the luciferase-polyA coding sequence from pGL3Basic (Promega, UK) and counter selection using 2-deoxy-galactose and glycerol. Correctly modified colonies were identified by PCR screening and pulsed-field gel electrophoresis.