hellbender_spleen
We collected tissues for RNA extraction from an eastern hellbender larva. This individual, part of a captive cohort at Purdue University, was euthanized after showing signs of failure to thrive. We euthanized the individual in a solution of MS-222 using an approved protocol by the Purdue University Animal Care and Use Committee (PACUC protocol # 1406001094). We immediately dissected the spleen and stored the sample in liquid nitrogen. We extracted RNA from the spleen tissue using TRIzol reagent (Invitrogen Corp., Carlsbad, CA) followed by the purification steps of the RNAeasy mini kit (Qiagen Co., Hilden, Germany). Sequencing libraries were made using the TruSeq Stranded RNA prep kit (Illumina Inc., San Diego, CA) which consists of poly-A RNA selection using oligo (dT) magnetic beads, fragmentation of RNA, and first- and second-round cDNA synthesis. Sequencing was performed using a HiSeq 2500 machine (Illumina Inc., San Diego, CA).