Zr duet dna rna miniprep plus kit

Embryos, larva, pupae, adult males and females were collected for later mRNA extraction. Tissue samples were mechanically homogenized using ZR Bashing Bead Lysis tube (ZymoResearch, CA, USA) with the FastPrep-24™ 5G Instrument (MP Biomedicals, Santa Ana, CA, USA). Nucleic acids were then extracted from homogenized suspension using the ZR-Duet DNA/RNA MiniPrep Plus kit (Catalog # D7003, ZymoResearch, CA, USA). Extracted RNA was quantified with RNA-specific fluorometric quantitation on a Qubit 2.0 Fluorometer using Qubit RNA HS Assay (Thermo Fisher Scientific, Waltham, MA, USA). Integrity of total RNA was assessed on an Agilent Bioanalyzer, using the RNA 6,000 Pico LabChip kit (Catalog # 5067-1513, Agilent Technologies, Santa Clara, CA).

Total RNA from crypts was isolated using QIAzol Lysis reagent (Qiagen) followed by isopropanol precipitation. RNA from Lgr5-eGFP^hi ISCs sorted by FACS were isolated using ZR-Duet™ DNA/RNA MiniPrep Plus Kit (Zymo Research) following the manufacturer’s instructions. Isolated RNA was quantified on Nanodrop 8000 (Thermo Fisher Scientific) and on Qubit 3.0 (Thermo Fisher Scientific). The quality of isolated RNA was analyzed by Fragment Analyzer (Agilent).

Postmortem tissue samples from elderly donors (20 controls, 20 AD patients), all exhibiting tauopathy (Braak stage I–IV controls and IV–VI AD, Table S1) [31 (link)] were obtained from the Newcastle Brain Tissue Resource (NBTR). All was approved by the joint Ethics Committee of Newcastle/North Tyneside Health Authority (following NBTR brain banking procedures) and Tel Aviv University Ethics Committee. Full written informed consent was obtained from tissue donors or their relatives where appropriate. All AD cases fulfilled the criteria for high AD neuropathological changes according to the National Institute on Aging-Alzheimer's Association (NIA-AA) guidelines. Tissues were homogenized using a Bullet Blender (Next Advance, Inc., NY). RNA and DNA were extracted from the same tissue with a ZR-Duet DNA-RNA MiniPrep Plus kit (Zymo Research, Irvine, CA).

Total RNA from crypts was isolated using QIAzol Lysis reagent (Qiagen) followed by isopropanol precipitation. RNA from Lgr5-eGFP^hi ISCs sorted by FACS were isolated using ZR-Duet™ DNA/RNA MiniPrep Plus Kit (Zymo Research) following the manufacturer’s instructions. Isolated RNA was quantified on Nanodrop 8000 (Thermo Fisher Scientific) and on Qubit 3.0 (Thermo Fisher Scientific). The quality of isolated RNA was analyzed by Fragment Analyzer (Agilent).

Forty postmortem olfactory bulb brain tissues and a similar number of hippocampus tissues (14 AD patients and 20 age matched non-demented controls) were obtained from Newcastle Brain Tissue Resource (NBTR) in accordance with the approval of the joint Ethics Committee of Newcastle and North Tyneside Health Authority and following NBTR brain banking procedures which includes full written informed consent from tissue donors or relatives. All AD cases fulfilled the criteria for high AD neuropathologic change according to the National Institute on Ageing-Alzheimer’s Association (NIA-AA) guidelines^{64 (link)}. Demographic data for the AD patients and age matched non-demented controls are presented in Supplementary Table 2. RNAs were extracted from these postmortem tissues following homogenization using Bullet Blender (Next Advance, Inc., NY, USA), and extraction with ZR-Duet DNA-RNA MiniPrep Plus kit (Zymo Research, Irvine, CA, USA). Next, RNAs were quantified as described above for LCLs. Supplementary Fig. 8 describes our study design and includes N values for each cohort.

RNA isolation was performed at GenXPro GmbH. Cell lysates were stored in liquid nitrogen before RNA isolation. Isolation of total RNA from ECs was performed using the ZR-Duet DNA/RNA MiniPrep Plus kit (Zymo Research, Irvine, CA, USA) according to manufacturer’s instructions. RNA samples were digested using DNase I and RNA integrity was accessed using automated capillary electrophoresis (RNA pico sensitivity assay, LabChip GX II Touch HT, Perkin Elmer, Villebon-sur-Yvette, France).