Illumina MiSeq sequencing
determination of transcript 3'-end
1 ��g of the total RNA instead of rRNA depleted RNA was ligated with 1 ��L of 150 ��M amino-blocked DNA adaptor (5��-p-NNAGATCGGAAGAGCGTCGTGT-3��) as described above. After ligation, rRNA was removed by using Hybridase�� Thermostable RNase H (Lucigen Corporation, Middleton, WI, USA). 13.5 ��L of the purified ligation product was mixed with 1.5 ��L of 10�� DNase I Reaction Buffer (New England BioLabs), 15 ��L of Hybridase complement buffer (90 mM Tris-HCl pH 7.5, 200 mM KCl), 1 ��L of anti-rRNA oligo mix (detailed composition is available in Figshare) and 2 ��L of 50 mM MgCl2 and incubated in thermal cycler (heat to 95 oC, cool down to 65 oC). 2 ��L of Hybridase�� Thermostable RNase H was added to the mixture and the mixture was incubated at 65 oC for 20 minutes, 90 oC for 1 second and 65 oC for 10 minutes. rRNA depletion reaction was cleaned up using RNA Clean & Concentrator Kits (Zymo Research, Irvine, CA, USA). The purified adaptor ligated RNA was then fragmented by incubating at 72 oC for 90 seconds in fragmentation buffer (Ambion, Inc, Austin, TX, USA). The fragmentation reaction was cleaned-up using Agencourt AMPure XP beads. The fragmented RNA (8 ��L in total) was reverse transcribed with SuperScript III Reverse Transcriptase using 1 ��L of 10 ��M reverse transcription primer (5��-TCTACACTCTTTCCCTACACGACGCTCTTC-3��) according to the manufacturer��s instructions. The cDNA was then purified with Agencourt AMPure XP beads. Another amino-blocked adaptor with different sequence (5��-p-NNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3��) was ligated to the cDNA with increased incubation time (8 h). The ligation product was purified using Agencourt AMPure XP beads and indexed by PCR with Phusion High-Fidelity DNA Polymerase using forward (5��-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT-3��) and reverse (5��-CAAGCAGAAGACGGCATACGAGATNNNNNN (6 nt index) GTGACTGGAGTTCAGAC-3��) primers. The PCR reaction was monitored using a CFX96 Real-Time PCR Detection System and stopped before the PCR reaction was fully saturated. The PCR product was purified with Agencourt AMPure XP beads.