title: Illumina MiSeq paired end sequencing
library_construction_protocol: PolyA RNA was isolated from 500 ng of input RNA using oligo(dT)25 Dynabeads (Invitrogen) by binding the RNA to the Dynabeads, collecting the beads with a magnetic rack, and removing the supernatant containing non-polyA RNA. The Dynabeads were washed in washing buffer then the mRNA was eluted in TE buffer. The binding and washing steps were repeated to remove remaining non-polyA RNA. After the binding and washing, the Dynabeads were resuspended in 2X Superscript III first-strand buffer with 10 mM DTT and incubated at 94 °C for 6 min to fragment the mRNA then immediately cooled on ice. The Dynabeads were collected on a magnetic stand and the fragment mRNA was moved to a new tube.  Fragmented mRNA in 2x RT buffer, random hexamers, and RNasin Plus was heated at 50 °C for 1 min to denature the RNA then placed immediately on ice. To this, water, Actinomycin D, DTT, dNTPs, and SuperScript III (Invitrogen) were added and reverse transcription was performed by incubating the mixture at 25 °C for 10 min then at 50 °C for 50 min.  RNAClean XP beads (Agencourt) were added to the mixture and incubated on ice for 15 min. The beads were collected on a magnetic stand, washed twice with 75 % ethanol then air-dried for 5 min. The RNA/cDNA hybrid was eluted in water.  A second strand reaction master mix including either 10x Blue Buffer or NEBuffer 2, dNTP (with dATP, dCTP, dGTP, and dUTP), RNase H, DNA polymerase I (Enzymatics), and water was added to each sample and incubated at 16 °C for 2.5 h.  Double-stranded DNA was purified using 1.8 volumes of AMPure XP beads (Agencourt) and eluted in water.  The DNA was dA-tailed by adding either 10x Blue Buffer or NEBuffer 2, dATP, water, and Klenow 3’-5’ exo (Enzymatics) and incubated at 37 °C for 30 min.  The dsDNA was purified using AMPure XP beads, and eluted in water. To each sample, a specific barcode adapter was added, along with Rapid Ligation Buffer and T4 DNA Ligase HC (Enzymatics). The Y-shape adapter ligation was performed by incubating at room temperature for 15 min.  DNA was purified and size selected by cleaning up with AMPure XP beads 3 consecutive times and eluting in TE buffer. The first cleanup was performed with 1 volume of AMPure XP beads, the second with 1.4 volumes, and the last with 1 volume incubated at room temperature for 5 min without washing to perform size selection. The supernatant was transferred to a tube with half of the new volume of AMPure XP beads and incubated at room temperature for 15 min. The beads were collected on a magnetic stand and washed twice with 75 % ethanol. The DNA was eluted in water.  The second strand of DNA was digested with Uracil DNA Glycosylase (Enzymatics) at 37 °C for 15 min.  The PCR reaction was set up containing UDG-digested DNA, primer A and B, Phusion HF Buffer, dNTP, water, and Phusion II (New England Biolabs). The reaction was incubated at 94 °C for 2 min, followed by 10-12 cycles of amplification (98 °C for 10 s, 65 °C for 30 s, 72 °C for 30 s). The DNA libraries were purified with 1.4 volumes of AMPure XP beads and eluted in TE buffer.