title: Amplicon sequencing of Lotus japonicus: hairy roots
design: To assess the efficiency of various CRISPR constructs in two rounds, DNA from Lotus japonicus roots was extracted using the CTAB method. Library preparation included a three-step PCR, to amplify target regions (producing amplicons LjLbdouble and LjLb3), add linker sequences and index sequences, respectively. Two libraries were made, the first one (run1) from samples transformed with a construct carrying the Cas9 gene under LjUB promoter, and the second (run2) from samples carring Cas9 gene under a 2x35S promoter (including some samples from the first round that were not sequenced during run1). After the performance of the in-house multiplex indexing and pooling of the PCR products per library, the sequencing-ready libraries were prepared with the KAPA Hyper Prep PCR Free kit (KAPA Biosystems, Wilmington, MA, USA), while sequencing was performed with the HiSeq 2500 instrument (Illumina, San Diego, CA, USA) at a Rapid Run mode generating 250 bp paired-end reads.