GSM6413421: PureLink Heart LPS sample #1; Mus musculus; RNA-Seq
1-2mg samples of frozen tissue were sampled with the CryoCore and put into separate wells of a 96 well plates.  100ul of either PIXUL PK Buffer, PureLink Lysis Buffer, or TRIZOL buffer was added to the wells and then all samples were sonicated in PIXUL sonicator (Matchstick Technologies, Inc Kirkland WA and Active Motif Carlsbad, CA). Samples are processed for 30 seconds in PIXUL with settings: Pulse=50, PRF=1.0 Burst=20.  TRIZOL samples then received additional 100ul of TRIZOL and followed manufacturers protocol for RNA isolation. PureLink samples then follow manufacturers protocol at step 1 of the 'Binding, Washing, and Elution' section of 'Purifying RNA from Animal Tissues' using on-column DNase digestion and a final elution volume of 20ul. PIXUL PK Buffer samples are immediately incubated (After sonication) in the same PIXUL plate at 95C for 20 minutes, and then put on ice for 5 minutes. Cooled samples are centrifuged in a plate centrifuge for 10 minutes at 4,200g and 4C and then put back on ice. 60ul of clear supernatant (without any floating residue) from each well is carefully transferred via pipette to a new semi-skirted 96 well PCR plate on ice. Isolation of nucleic acids with 1.8x SPRI beads is done as per the Omega Bio-tek protocol (Norcross, GA) with a two-minute final SPRI bead drying time and final elution to 50ul of ultrapure ddH2O and then put samples on ice. Finally, DNase I and RNAse Out are added to the eluted RNA samples and DNase I digestion is done as per the manufacturer's protocol. Sequencing libraries were prepared using Zymo-Seq RiboFree Total RNA Library Kit with starting RNA between 205-480ng and libraries amplified between 13-14 cycles of PCR as per manufacturers protocol. Libraries were diluted as per Illumina NextSeq2000 Sequencing System Guide to a final pooled loading concentration of 650pM in resuspension buffer (RSB) plus Tween 20 with a 10% PhiX spike-in and sequenced in Illumina P2 cartridges on NextSeq 2000 that employed a dual-index, paired-end, 75 base read length (PE75).