GSM2232751: A229T4-ISC_SPENrep1; Drosophila melanogaster; RNA-Seq
Gut dissociation step was performed as described in (Dutta et al., 2013) using PBS Elastase (final concentration 1mg/ml) solution and mechanic pipetting. Dissociated cells were resuspended in PBS, 1% BSA and FACS sorted (FACS Aria BD Biosciences) using the following parametters : 20psi, 100µm nozzle. Diploid RFP+ positive fractions (ISCs) were separated from the diploid RFP+ GFP+ double positive fractions (EBs). Cells were directly collected in TRIzol (Life Technologies). RNAs was extracted by performing two series of Phenol-Chloroform extractions at 4°C, followed by DNase treatment according to the manufacturer’s instructions. Around 100ng total RNA were collected from 100 000 sorted cells. Total RNA quality and quantification were assessed on an Agilent Bioanalyzer using the Agilent RNA 6000 nano kit. A classical Illumina TruSeq Stranded RNA (LS) protocol was used to build the libraries, from poly(A)+-enriched mRNA (by Ribo-Zero rRNA removal Illumina kit). Three biological replicates were sequenced per conditions on the Illumina HiSeq 2500.