GSM6965729: HA-BFD2_stress_IP; Toxoplasma gondii; RIP-Seq
At the time of harvest, monolayers were scraped and serially extruded through 27-and 30-gauge needles to release intracellular parasites. Parasites were then isolated from host cell debris by additional passaging through a Millex filter unit (5 μm pore size, Merck Millipore), pelleted by centrifugation, and resuspended in MAGNA-RIP lysis buffer. For an 'input' sample, 10% of the starting lysate was set aside. The remaining 90% was subjected to IP with mouse anti-HA antibodies (clone 16B12, Abcam) using the MAGNA-RIP kit (Millipore) as described in the product technical manual. Total RNA was isolated in parallel from both enriched (IP) and unenriched (input) samples by canonical phenol:chloroform extraction. Libraries for RNA-seq were prepared using the Swift Low-Input Library Prep Kit with poly(A) enrichment following manufacturer's protocols.