GSM5649963: PBS- and LNP- treated murine liver cells; Mus musculus; OTHER
Mice were sacrificed 1 day after administration of LNPs and immediately perfused with 20 mL of 1X PBS through the right atrium. The liver was isolated immediately following perfusion, minced with scissors, and then placed in a digestive enzyme solution with Collagenase Type I (Sigma Aldrich), Collagenase XI (Sigma Aldrich), and Hyaluronidase (Sigma Aldrich) at 37oC and 750 rpm for 45 minutes. Digested tissues were passed through a 70 μm filter and red blood cells were lysed. Cells were stained to identify specific cell populations and sorted using a BD FacsFusion cell sorter. To allow for pooling of samples into a single device, cells were stained with a streptavidin conjugated H-2 MHC class I (M1/42, BioLegend) antibody and biotinylated cell hash oligos were added at 0.5 uM final concentration after a single wash to remove unbound antibody. Pooled microwell-seq barcoded beads were placed in a reverse transcription reaction containing 200 U M-MLV Reverse Transcriptase (Biochain Institute), 1x RT buffer, 20 U RNAse inhibitor (NEB), 1 M betine (Sigma), 6 mM MgCl2 (Sigma), 2.5 mM DTT (Thermo Fisher), 1mM dNTP (NEB), and 1 uM TSO primer. The beads were incubated for 90 minutes at 42 C for 90 minutes followed by a hold 4 C with constant shaking at 500 RPM. After the reverse transcriptase step, enzyme was removed with a wash of 1 x TE with 0.5% SDS , followed by a 1 x TE with 0.01% Tween 20 and finally a wash in 100 mM Tris-HCl pH 8.0. To remove any unused single stranded oligo from the beads they were treated with 200 U of Exonuclease I (NEB) in 1x ExoI buffer for 60 minutes at 37 C with 500 RPM shaking. Following the digestion, excess ExoI was removed using the previously described TE-SDS, TE-Tween 20 and Tris-HCL pH 8.0 washes. After removal of ExoI, the beads were resuspended in 200 ul of Platinum II hot-start master mix (Thermo Fisher) with IS-PCR, p7 Multi Barcode Rvs and Hash p7 Rvs primers and the first round PCR was performed using the following cycling conditions: one cycle at 94 C for 2 minutes, 12 cycles of 94 C for 15 seconds, 60 C for 15 seconds and 68 C for 2 minutes. The sample was pooled, the beads were removed and discarded, and the sample was purified using 0.6x SPRI beads. The long RNA fragments were collected on the SPRI beads, while the shorter barcode and hash reads remained in the PCR supernatant, these were subsequently purified using 2.0x SPRI beads and saved for use during the final round PCR. The RNA sample was then treated with TN5 transposase to fragment and add on sequencing handles for PCR. Both the DNA and fragmented RNA sample was then amplified using a final round of PCR with non-hot start Q5 high fidelity polymerase (NEB), P7 Nextera index adapters and the Microwell P5 primer using the following cycling conditions: one cycle at 70 C for 5 minutes, 12 cycles of 98 C for 30 seconds, 58 C for 30 secconds and 72 C for 90 seconds with a final extension at 72 C for 2 minutes. The samples were then purified using 0.8x SPRI beads and pooled at a 10:1 molar ratio of RNA to DNA and sequenced on a Illumina HiSeq paired-end 150 cycle run.