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
Entamoeba     histolytica     HM1:IMSS     is     a     virulent     strain     isolated     in     1967     from     a     colonic     biopsy     of     rectal     ulcer     from     adult     human     male     with     amoebic     dysentery,     Mexico     City,     Mexico.     The    HM1-­‐IMSS    was    deposited    in    the    American    strain    collection    (ATCCH    30459TM)    and    it    is    a     gift     of     Professor     Ruy     Perez     Tamayo     (UNAM,     Mexico).     To     maintain     virulence,     the     HM1-­‐IMSS     strain    has    been    passed    through    the    liver    of    hamsters    (roughly    180    passages    since    isolation).     The    axenic    Entamoeba    histolytica    strain    HM1:IMSS    was    cultured    in    TYI-­‐S-­‐33    medium    at    37°C     (28).     The     fresh     liver     isolate     was     maintained     in     culture     for     16     hours,     for     two     weeks     or     non-­‐ more    of    three    months    until    the    experiments    were    done.    Attenuated    trophozoites    were    derived     from    the    HM1-­‐IMSS    virulent    strain    by    prolonged    axenic    culture    (more    than    10    years)    and    have     lost    the    ability    to    form    liver    abscesses. 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.