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
Four-­‐week-­‐old     male     Syrian     golden     hamsters     (Mesocricetus     auratus),     with     a     weight     ranging     from     90     to     100g     were     inoculated     by     the     intraportal     route     with     10 6      wild-­‐type     HM1:IMSS    trophozoites.    Treatment    of    animals    and    surgical    procedures    were    done    according     to    published    procedure   and    with    agreement    of    Pasteur    Institute    rules    for    animal    infection.     From     7     days     post-­‐infection,     hamsters     were     sacriJiced,     livers     removed.     For     RNASeq     experiments,    pieces    of    infected    liver    were    incubated    in    TY1-­‐S-­‐33    medium    and    parasites    were     allowed    to    adhere    to    the    culture    tube    for    16    hours.    Liver    tissues    were    then    discarded    and    the     pool    of    trophozoites    used    to    purify    RNA.     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.