Illumina MiSeq sequencing
determination of transcript 3'-end
The equal amounts of DNase I-treated RNA from 4 time points were mixed and used for the input of Term-Seq library construction. The RNA was treated with Ribo-Zero rRNA Removal Kit Bacteria (Epicentre) to deplete rRNA. The resulting 500~900 ng of rRNA-depleted RNA was mixed with 1 ��L of 150 ��M amino-blocked DNA adaptor (5��-p-NNAGATCGGAAGAGCGTCGTGT-3��), 2.5 ��L of 10�� T4 RNA ligase 1 buffer, 2.5 ��L of 10 mM ATP, 2 ��L of DMSO, 9.5 ��L of 50% PEG8000, and 2.5 ��L of T4 RNA ligase 1 (New England BioLabs). The mixture was incubated at 23 oC for 2.5 hrs and reaction was cleaned-up using Agencourt AMPure XP beads (Beckman Coulter). The 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 (Invitrogen) 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 as the above with increased incubation time (8 hrs). 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 (Bio-Rad Laboratories, Hercules, CA, USA) and stopped before the PCR reaction was fully saturated. The PCR product was purified with Agencourt AMPure XP beads.