GSM4083096: Chick_Nematode_Treated; Ascaridia galli; RNA-Seq
Preparations of Aqueous extract (Maceration method) Bryophyte, Pottia lanceolata samples were cleaned removing soil debris, by thorough washings with water. The active ingredient was extracted, using cold maceration method dried in shade, powdered using mortar and pestle. The crude aqueous extract was centrifuged, at 5,000 rpm for 5 minutes, using Remi centrifuge. The clear supernatant was used for the treatment and the precipitate was discarded. Piperazine citrate (glaxo smithkline) was used as the standard anthelmintic drug during the experimentation. To study the gene expression pattern, the control and treated (bryophyte extract Pottia lanceolata) parasites were preserved in RNA later solution, and were subjected to RNA extraction and isolation using RNA mini easy kit. The Extraction was performed as per the protocol, provided by the manufacturer. Extracted RNA was subjected to quality and quantity check, prior to library construction. Library preparation was performed following NEB Next Ultra RNA Library Prep Kit (#E7490L). In brief, approximately 110-350 ng of Total RNA was used to isolate mRNA using Poly dT beads. mRNA was fragmented and converted to cDNA according to the protocol. cDNA was end repaired and purified using Ampure XP beads. Cleaned DNA was adapter ligated and purified using Ampure XP beads. These adapter ligated fragments were subjected to 12cycles of PCR using primers provided in the kit. The PCR products were purified using Ampure XP beads. Quantification and size distribution of the prepared library was determined using Qubit fluorometer (Table 1) and Agilent Tape station D1000 Kit (Agilent Technologies) according to the manufacturer's instructions. The steps followed to generate the FastQ reads from Total RNA libraries are as follows: 1. The pooled/multiplexed libraries were put into the Illumina Hiseq Sequencer for sequencing which generated raw data in the bcl format. 2. Bcl files were De-multiplexed using sample wise Barcode information to get the sample specific sequences. 3. De-multiplexing resulted into raw reads in FastQ format, which was used for downstream analysis.