Illumina Genome Analyzer II sequencing; small RNA sequencing_20 oct 2013
small RNA sequencing_20 oct 2013
An. stephensi from laboratory stock were reared under controlled conditions at 28 + 2degreesC, 70 -75% humidity.  Adult mosquitoes were fed with 2% sterile glucose solution and water soaked raisin.Samples were prepared from An. stephensi at 5 different conditions. 5-6 days old sugar fed adult female mosquitoes were collected. Blood fed and parasite infected mosquitoes were collected at two time points, at 42 hours and five days post blood feeding. Whole body of all mosquito samples were stored in Trizol (Invitrogen) until RNA extraction. Stock solution of Plasmodium vinckei petteri 279 BY was thawed and injected into 5 weeks old BALB/C mice. When gametocytemia reached 0.05%, naive 4-5 days old female mosquitoes were fed on infected mice. Midguts of mosquitoes on 5th day post infection (dpi) were dissected, stained in mercurochrome and observed for the presence of oocysts under microscope.  Presence of oocysts confirmed infection of mosquitoes by Plasmodium parasite.  BF 42h and BF 5d mosquitoes were fed on normal mice blood and were collected at specified time points. Total RNA enriched in small RNA population was extracted using miRNeasy kit (Qiagen) as per the manualﾒs protocol. Quality and quantity of RNA was checked by using Agilent 2100 Bioanalyzer RNA Nano 6000 kit. Illumina Truseq small RNA libraries were made as per the manufacturer's instructions (Illumina Inc). 1ug of total RNA was ligated with 3prime and 5prime adaptors followed by reverse transcription using RT primers. Following PCR amplification of the adaptor enriched fragments, population of small RNA within length 140-160 bps were ran and eluted from 6 % TBE PAGE gel. Eluted product was precipitated using sodium acetate and ethanol and was dissolved in RNAase free water. These RNA samples were sent for high throughput sequencing using Illumina Genome Analyzer II.
ICGEB AT NEW DELHI
E-MTAB-2045:assay 2
ICGEB at New Delhi
E-MTAB-2045:assay 2
Experimental Factor: growth condition
uninfected mouse blood
Experimental Factor: time
42 hour