design: To select ribozymes with improved trans-splicing efficiency, in vitro trans-splicing reactions were performed under near-physiological conditions. Five independent reactions (30 L each) were prepared, each containing 100 nM ribozyme library and 100 nM JAK2-V617F mRNA substrate in a physiological reaction buffer (5 mM MgCl, 135 mM KCl, 50 mM MOPS/NaOH pH 7.0, 20 M GTP, and 2 mM spermidine) and incubated at 37 C for one hour. Ribozymes were pre-incubated for 5 minutes at 37 C in the presence of MgCl to promote proper folding before substrate addition. Reactions were quenched using 4 L of 50 mM EDTA, and products were ethanol precipitated, and reverse transcribed using Superscript III reverse transcriptase (Invitrogen, Waltham, MA, USA). The resulting cDNA was PCR-amplified and subjected to two additional rounds of PCR that added Illumina adapters and Illumina barcodes (Illumina, San Diego, CA, USA). The final product was submitted for HTS using Illumina Novaseq 6000 (UC San Diego IGM Genomics Center).