GSM7140520: ACL repair 2, synovium, 52 weeks; Sus scrofa; RNA-Seq
Cartilage specimens were homogenized in 2 ml tubes (MP Biomedical) containing 500 ml of frozen TRIzol (Life Technologies) using a sterile drill bit while tubes were submerged in liquid nitrogen. After one round of drilling, tubes received an additional 500 ml of liquid TRIzol, were flash frozen in liquid nitrogen, and again homogenized using the drill bit. Total RNA was extracted using phenol-chloroform and purified using PureLink RNA Mini Kit (Life Technologies). Synovium specimens were immediately placed in 2 ml lysing matrix S tubes (MP Biomedicals, Santa Ana, CA) and homogenized in 1 ml TRIzol (Life Technologies, Carlsbad,CA) using a Fast Prep-24 Instrument (MP Biomedical). Samples were then flash frozen in liquid nitrogen, transferred to dry ice, and stored at -80 ̊C until RNA isolation. Total RNA was extracted using phenol-chloroform and purified using PureLink RNA Mini Kit (Life Technologies). All cartilage and synovium RNA isolates were then library-prepped using KAPA mRNA HyperPrep with RiboErase (Roche) and subsequently pooled and sequenced together on a NovaSeq 6000 S2 Flow Cell with 100-bp paired-end reads (Biopolymers Facility, Harvard Medical School, Boston, MA).