GSM3666772: 3Dmodel of the human intestine in the presence of Entamoeba histolytica [A3]; Homo sapiens; Entamoeba histolytica HM-1:IMSS; RNA-Seq
1ml of Trizol reagent (Qiagen) was added to the sample and incubated for 5 min at room temperature, then 200 µl of chloroform were added. After 2 min incubation at room temperature, the samples were spin down at 12000 x g, 15 min at 4°c. The supernatant was complete with 70% ethanol (V/V) and then transferred to a RNeasy Mini spin column (Qiagen) and the RNA was recovered according to the Qiagen' protocol. DNase treated RNA molecules isolated from - description of conditions - were treated for library preparation using the TruSeq Stranded mRNA sample preparation kit (Illumina, San Diego, California) according to manufacturer's instructions. An initial poly(A) RNA isolation step (included in the Illumina protocol) is performed on n ng of total RNA to keep only the polyadenylated RNA fraction and remove the ribosomal RNA. A step of fragmentation is then performed on the enriched fraction, by divalent ions at high temperature. The fragmented RNA samples were randomly primed for reverse transcription followed by second-strand synthesis to create double-stranded cDNA fragments. No end repair step was necessary. An adenine was added to the 3'-end and specific Illumina adapters were ligated. Ligation products were submitted to PCR amplification. The obtained oriented libraries were controlled by Bioanalyzer DNA1000 Chips (Agilent, # 5067-1504) and quantified by spectrofluorimetry (Quant-iT™ High-Sensitivity DNA Assay Kit, #Q33120, Invitrogen). Sequencing was performed on the Illumina Hiseq2500 platform to generate single-end 65 bp reads bearing strand specificity.
GEO
GSM3666772