GSM5050461: Library 2 with Polylinker Rep1 RNA; Mus musculus; OTHER
Target library reporter sequences were amplified using primers 'GTGGAGCAGTACGAGCG' and 'TAGCCAGAAGTCAGATGCTCAAG' for 30 cycles (NEB Q5) at an annealing temperature of 67C and then purified with the Monarch PCR kit. Illumina adaptors were then added via two further rounds of PCR. First, custom P1 indexing barcodes were tailed on for 5 cycles at an annealing temperature of 55C followed by 5 cycles with no annealing step (NEB Q5). PE2 indexing barcodes were then tailed on by amplifying 2 microliters of the previous reaction for 5 cycles at an annealing temperature of 66C followed by 5 cycles with no annealing step (NEB Q5) and then purified 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.