Fig. 7.

Identification of optimal library construction chemistry and ligation condition. a Workflows of three categories of library construction chemistries. Work flow-A has cleanups after every step of library construction and in Work flow-B the cleanup after A-addition is removed where as in the most-streamlined Work flow-C end-repair and A-addition are coupled into one reaction and the cleanup after A-addition is removed. b Comparison of library yield between the three NEB workflows. PCR-free libraries were generated using the chemistries that represented the workflows depicted in (a) using two different amounts of gDNA as inputs. qPCR was applied to measure the final library yield. c Optimization of ligation. For the best performing chemistry (NEB workflow-B), ligation time point analysis was performed by varying the adapter amount. This was performed using our ssRNA-seq pipeline. n = 3; error bars = Standard Deviation