title: GSM5050454: Library 2 with Rho Rep2 RNA; Mus musculus; OTHER
library_construction_protocol: Target library reporter sequences were amplified using primers 'CACCTGTTCCTGTAGGCATGC' and 'TATTACAATTGTAGCCAGAAGTCAGATGCTCAAG' for 6 cycles at an annealing temperature of 66C followed by 18 cycles with no annealing step (NEB Phusion) and then purified with the Monarch PCR kit (NEB). PCR amplicons were digested using MfeI-HF and SphI-HF (NEB) and ligated to custom annealed adaptors with PE2 indexing barcodes and phased P1 barcodes. The final enrichment PCR used primers 'AATGATACGGCGACCACCGAG' and 'CAAGCAGAAGACGGCATACGA' for 20 cycles at an annealing temperature of 66C (NEB Phusion), followed by purification with the Monarch PCR kit. Samples were mixed at equal concentration and sequenced on the Illumina NextSeq platform. RNA was extracted from TRIzol according to manufacturer instructions and treated with TURBO DNase (Invitrogen). cDNA was prepared using SuperScript RT III (Invitrogen) with oligo dT primers.