Small RNA seq of FACS sorted progenitor cells in Drosophila Intestine
Small RNA seq of FACS sorted progenitor cells in Drosophila Intestine
The guts were digested with Collagenase-IV (10mg/ml in 1XPBS-DEPC). Incubate the guts at 27ºC for 1 hour with slow agitation. The guts were agitated every 15 min with pipette for 30-40 times for full dissociation. The dissociated cells were centrifuges at 300rpm at 4oC for 20 mins. The pellet containing the cells were resuspended in ice cold PBS-DEPC and processed for FACS analysis with 1 µl of propidium iodide. The cells were sorted based on GFP fluorescence and size using the BD FASC Aria II system.  Total RNA was extracted from sorted GFP positive RFP negative cells using mirVanaTM miRNA Isolation kit (Invitrogen) according to manufactures protocol Total RNA was extracted from sorted GFP positive RFP negative cells using mirVanaTM miRNA Isolation kit (Invitrogen) according to manufactures protocol. miRNA seq analysis was performed at UR Genomics Research center. The total RNA concentration was determined with Nanodrop 1000 spectrophotometer (Nanodrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). 500 ng of Total RNA was utilized as the input for sequencing library construction with the NEB Next small RNA library prep kit (New England Biolabs), following the manufacturer's protocol. Importantly, 3' and 5' adapters were diluted 2-fold for ligation steps, as specified by the manufacturer. Following 3'adaptor ligation, RT primer incubation, and 5' adapter ligation, cDNA was synthesized, and final library amplification was performed with 12 cycles of PCR. Library quantity and quality were assessed using a Qubit fluorometer (Thermofisher) and Tapestation 2200 (Agilent), respectively. Sample libraries were proportionally pooled and isolated for the PCR reaction mix using the Qiagen MinElute PCR Reaction Cleanup kit and Library fragments between 125-160bp were size selected using a 3% cassette on a PippinHT (Sage Science). The size selected library pool was then sequenced on an illumina HiSeq 2500, generating single end reads of 50nt.