GSM5454446: Template switch library from STARR-seq GP5d binary RNA, replicate 1; Homo sapiens; OTHER
Total RNA was isolated Qiagen RNeasy maxi kit after 24 hours of transfection. Input library for random promoter-enhancer binary STARR-seq was constructed as described in Arnold et al. Science 2013. For generating a sequencing library using a template switch strategy, a 40-ug aliquot of isolated total RNA from the random promoter-random enhancer STARR-seq experiment in GP5d cell line was used. Briefly, TurboDNase-treated RNA was incubated at 72 C for 3 min with a custom biotinylated STARR-seq specific RT-primer and dNTPs in two reactions having 25 ng of RNA per reaction, followed by immediate chill on ice. The first strand synthesis was performed using 100 units of Superscript IV RT (Invitrogen) in 1x SS-IV RT buffer containing 10 units of RNase OUT (Invitrogen), 5 mM DTT, 6 mM MgCl2 (Sigma), 1M Betaine (Sigma) and 1 uM custom template switch oligo compatible with the custom CpG-free Illumina sequencing by incubating the reactions at 50 C for 15 min followed by 80 C for 10 min. cDNA was purified using AMPure XP beads and one cDNA reaction product was split into two for PCR amplification using KAPA HiFi master mix (Roche) together with USER enzyme (NEB) and for enrichment of STARR-seq reporter-specific template. The PCR product was purified using AMPure XP beads followed by a second PCR for a total of 5 cycles for Illumina library preparation using custom CpG-free oligos. Illumina sequencing strategy adapted for  pCpG-free STARR-seq construct for random promoter-enhancer and template switch library using custom adapters, sequencing and index primers.