Illumina HiSeq 2000 sequencing; Extensive transcriptome analysis identifies essential genes sustaining intestinal invasion by Entamoeba histolytica
Extensive transcriptome analysis identifies essential genes sustaining intestinal invasion by Entamoeba histolytica
1.6X10 5     trophozoites     were     added     to     the     luminal     face     of     the     colon     and     incubated     in     Krebs     buffer     at     37°C     for     1     and     7     hours.     After     1     hour     of     incubation,     mucus     interacting     trophozoites     were     collected     by     pipetting     the     mucus     layer     for     RNA     puri?ication.     Tissues     (3     independent     samples)     were     processed     in     accordance     with     the     French    government’s    guidelines    for    research    on    human    tissues    and    the    French    Bioethics    Act.     The     applied     protocol     was     approved     by     a     regional     investigational     review     board     (Comité     de     Protection    des    Personnes,    Ile    de    France    VII,    Paris,    France)    and    an    institutional    review    board     (Institut    Pasteur    Recherche    Biomedicale,    Paris,    France;    reference    RBM/2009.50). Trophozoites     of     E.     histolytica     (approximately     1X10 6 )     were     harvested.     Total     RNA     was     extracted     from    these    trophozoites    using    Trizol    reagents    (Invitrogen,    Saint    Aubin,    France).    10    μg    of    total     RNA     have     been     polyA     enriched     twice     using     Sera-­‐Mag     Magnetic     Particles     Oligo(dT)     coated     (Thermo     ScientiOic,     #28152103011150).     The     samples     were     Oinally     eluted     in     10,5μL     DNase,     RNase-­‐free    water.    Libraries    were    constructed    using    ScriptSeq    mRNA-­‐Seq    Library    Preparation     Kit     (Epicentre,     #SS10924)     following     manufacturer's     recommendations.     Libraries     have     been     puriOied     after     PCR     using     double     Solid     Phase     Reversible     Immobilization     (dSPRI)     similarly     to     previously     described     protocol.     Libraries     were     controlled     by     Agilent     Bioanalyzer     DNA     High     Sensitivity    Chips.