Next generation sequencing

STAT5a cDNA was cloned into the pTracer-EF/V5-His mammalian expression vector (v. C, #V88720, Thermo Fisher Scientific) in sequence with the C-terminal V5 tag. Site-directed mutagenesis was performed using either Phusion Site-Directed Mutagenesis kit (for non-GC rich areas of the STAT5a sequence; ie, Y694F and S726A; cat #F541, Thermo Fisher Scientific) or Q5 Site-Directed Mutagenesis kit (for GC rich areas of the STAT5a sequence; ie, S780A; cat #E0552S, New England Biolabs, Ipswich, MA). Construct sequences were confirmed by next generation sequencing (Eurofins Genomics, Louisville, KY). STAT5a-V5 constructs were transfected with 1.5 μg pMDG plasmid and 6 μg pCMVΔR8.91 into HEK293T cells (ATCC) using Lipofectamine 3000. 48 h post-transfection, lentiviral supernatant was collected. MCF-7 cells were infected with lentiviral supernatant supplemented with fresh growth media and 8 μg/ml polybrene by spinfection at 500 g for 2 h at 32 °C. Infected cells were selected by Zeocin (cat #R25001, Thermo Fisher Scientific) 48 h post infection.

Next generation sequencing (Eurofins MWG Operon, Germany) of cDNA libraries prepared from MSG and PSG was performed. Briefly, poly(A) RNA was primed with random hexamers and was reverse transcribed for first strand synthesis. Then two unique adapters, A and B were ligated on 5′ and 3′ ends of the cDNA respectively and amplified with primers containing first four phosphorothioate-modified bases and 5′ biotinylated B-primer using a proofreading DNA polymerase. Reassociated ds-cDNA was separated from ss-cDNA by hydroxylapatite chromatography, and ss-cDNA was amplified for normalization. Library preparation and GS FLX Titanium sequencing of cDNA in the size range of 500–700 was done to obtain raw reads which were assembled to contigs. The contigs so obtained were merged with EST library sequences of A. assama PSG and MSG tissues from WildSilkBase and standalone BLASTn was performed using full length A. pernyi H-fibroin gene sequence as a query under general algorithmic parameters15 (link). The specificity of the positive hits was checked through pairwise alignment with query sequences on CLUSTAL Omega program and by conceptual translation of the positive sequences47 .

The PCR product was subjected to Next-Generation Sequencing by Eurofins Genomics. Sequences were generated with a MiSeq system using a 2x250 paired-end module. A single run was conducted which yielded 20.95 million reads and 5.24 gigabasepairs of data. The percentage of reads with Q score above 30 was 76.19% and the mean Q score was 30.59. The pair-end reads were combined using the FLASH program [24 (link)]. The result was a set of 8,573,790 individual nucleotide sequences (about 82% yield). Sequences were translated and converted to fasta format using Matlab with the BioInformatics toolbox (Mathworks). Searches within this database were carried out using BLAST (National Library of Medicine).