RNA extracted from MCF10A. Cells were purchased from ATCC and cultured as indicated
Total RNA is isolated using Tri-reagent (Ambion) and DNase-treated (Ambion). An oligo(dT) primer containing a VN-anker, a uridine, the sequence of the sequencing adapter and biotin (/5BiosG/CAGACGTGTGCTCTTCCGATCTTTTTTTTrUTTTTTTTTVN) is attached to streptavidin-coated magnetic beads (M280, Invitrogen). Total RNA is incubated for 5 min at 65°C, followed by incubation with the coated magnetic beads for 10 min at 45°C. First strand synthesis is carried out using Superscript III reverse transcriptase (Invitrogen) at 50°C. The enzyme is inactivated by incubation at 70°C for 15 min. Second strand synthesis is carried out using Second stand synthesis buffer (Invitrogen), dNTPs, DNA polymerase I (NEB), E. coli ligase (NEB) and RNase H (Invitrogen). To exchange the buffers and to remove enzymes from the previous step, we treat the samples with Pronase (Roche). We introduce a nick at the position of the uridine with RNAse HII (NEB). Then, nick translation is carried out with DNA polymerase I (NEB) and dNTPs for 8 min at 8°C. The reaction is stopped by the addition of  EDTA. At the new position of the nick we create a blunt end using T7 exonuclease, mung bean nuclease (NEB) and Klenow enzyme (NEB). Previously annealed Illumina truseq sequencing adapters (Ad1: 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT; Ad2: 5'- AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTAT) are then blunt- end ligated. The magnetic beads are suspended in 39 ul H2O. 5 ul of beads are used in a PCR reaction. 10-13 cycles of PCR are carried out with Phusion polymerase (NEB), a forward primer (5'-AATGATACGGCGACCACCGAGATC) and one of the Illumina truseq barcode reverse primers. For preparation of the library 6 PCR reactions are performed, run on 8% TBE gels (Invitrogen) and the smear between 160 bp and 220 bp is cut out, gel-extracted, ethanol-precipitated, analyzed by Bioanalyzer (Agilent) and sequenced on Illumina Hi-seq machines. A detailed protocol of 3'-seq will be available at the author's webpage.