GSM5918695: 12nucRNAPos; Mus musculus; RNA-Seq
The purification of viable, cell-specific nuclei from brain tissue from Tam-induced Aldh1l1-cre/ERT2+; NuTRAP+ mice was achieved by combining two previously published protocols, with modifications32,37,38. For each sample, two retinae were homogenized in 1 mL ice-cold nuclei EZ lysis buffer (#NUC-101, Millipore Sigma) supplemented with 1X Halt protease inhibitor cocktail (ThermoFisher Scientific) and 1 mM DTT using a glass dounce tissue grinder set (20 times with pestle A and 20 times with pestle B). Undissociated tissue, largely composed of blood vessels, was removed by centrifugation at 200 x g for 1.5 min at 4°C, and the supernatant containing the nuclear material filtered through a 30 µm strainer and centrifuged at 500 x g for 5 min at 4°C. The resulting nuclear pellet was resuspended in nuclei lysis EZ buffer, incubated on ice for 5 min, washed by centrifugation, and resuspended in 200 µL nuclei EZ storage buffer by gentle trituration with a micropipette. From the total resuspended pellet volume, 10% was reserved as input nuclei fraction and the rest was diluted with 1.6 mL nuclei purification buffer (NPB: 20 mM HEPES, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 1X Halt protease inhibitor cocktail, 1mM DTT), and subjected to the INTACT protocol. Briefly, 30 µL of resuspended M-280 Streptavidin Dynabeads (#11205, ThermoFisher Scientific) were added into a fresh 2 mL microcentrifuge tube and washed with 1 mL of NPB using a DynaMag-2 magnet (#12321; ThermoFisher Scientific) for a total of three washes (1 min incubation/each). The washed beads were reconstituted to their initial volume (30 µL) with NPB and gently mixed with the nuclear suspension. The mixture of nuclei and magnetic beads was incubated at 4°C for 40 min under gentle rotation settings to allow the affinity binding of streptavidin beads to the cell-specific, biotinylated nuclei. After incubation, the streptavidin-bound nuclei were magnetically separated with the DynaMag-2 magnet for a period of 3 min. The nuclei bound to the beads were washed in the magnet for three washes (1 min/each), resuspended in 30 µL of NPB, and reserved as the positive nuclei fraction. From the nuclear fraction [input and positive (enriched in biotinylated nuclei)], an aliquot was mixed with equal volume of DAPI counterstain and used for confocal microscopy visualization. The AllPrep DNA/RNA kit Micro (#80284, Qiagen, Germantown, MD) was used to extract gDNA and nuclear RNA from each sample. gDNA and nuclear RNA were quantified with a Nanodrop One spectrophotometer (Thermofisher Scientific) and its quality assessed by genomic DNA D1000 (#5067-5582) and HSRNA ScreenTape, respectively, with a 4150 TapeStation analyzer. The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used on 25 ng of total RNA for the preparation of strand-specific sequencing libraries from each TRAP-isolated RNA sample (input and positive fraction) and from conventionally isolated RNA samples from brain (tissue), according to manufacturer's instructions. Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, the addition of a single 'A' base, and finally ligation of adapters. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HS RNA ScreenTape (#5067-5579; Agilent Technologies).