Tapestation rna analysis screentape

A pUCIDT vector (IDT) was used to generate a GFP construct driven by an SP6 promoter, with a short 5′UTR (48nt), a 3′UTR (60nt) that contained the two universal adaptors sequences (40nt) as a cloning site, and a 36nt poly(A) (Figure 1A). The synthetic oligonucleotide library was amplified via the universal adaptors sequences and cloned it into the 3′UTR via two fragments Gibson assembly (NEB) with the cloning site adaptors. Bacterial transformations were performend by electroporation into Endura electrocompetent cells (Lucigen) according to the manufacturer's recommendations, and in large enough volumes and efficiencies to ensure at least 10× coverage of the oligonucleotide library. All colonies grown overnight from transformation were harvested and plasmids extracted with EZNA Plasmid Mini (Omega). Plasmid library was linearized by enzymatic restriction at the end of its 3′UTR, either before (A- library) or after (A+ library) the poly(A) sequence. mMessage mMachine Sp6 kit (Thermo Fisher) was used to in-vitro transcribe a library of mRNA reporters from linearized plasmids. In-vitro transcribed mRNAs were fully processed, capped and without any introns. Resulting mRNA levels were quantified by Qubit fluorometric quantification (Thermo Fisher) and length validated by Tapestation RNA analysis screen tape (Agilent).

Smart-seq was performed as described previously (28 (link), 58 (link)). Briefly, total RNA was isolated from naïve and activated B cells with Trizol (Thermo Fisher Scientific), and the integrity of the RNA was accessed with TapeStation RNA analysis ScreenTape or BioAnalyzer RNA Pico Kit (Agilent). Ten nanograms of RNA was reverse-transcribed using oligo-dT₃₀ VN primer in the presence of Template Switching Oligo (TSO) with SuperScript II reverse transcriptase. cDNA was pre-amplified with IS PCR primers, and PCR products were cleaned up with Ampure XP beads. One nanogram of the PCR product was used to generate library using Nextera XT Library Prep Kit (Illumina), and tagmentated DNA was amplified for a 12-cycle PCR and purified with Ampure XP beads. Libraries were sequenced on an Illumina HiSeq 2500 with single-end 50-bp reads.