Reagents were obtained from the following sources. Antibodies: b-actin (8457), FTH1 (3998), IRP1 (20272), IRP2 (37135), S6 (2217), and TFR1 (CD71, 13208) were from Cell Signaling Technologies; NFS1 (sc-365308), ISCU (sc-271468), and FECH (sc-377377) were from Santa Cruz Biotechnology; and FXN (ab113691) and FTL (ab218400) were from Abcam. Custom-made NRF2 antibody was provided by E. Schmidt, Montana State University. Cells: BT549, HCC1954, HCC70, HEK-293, Hs578t, MCF7, and MDA-MB-231 were from American Type Culture Collection; EFM19 was from DSMZ. Chemicals: RPMI (MT10040CV) was from Corning; PrimeStar HS DNA Polymerase Premix (R040A) was from Takara; RNeasy Plus Mini Kit (74136), Qiaprep Spin Miniprep Kit (27106), and QIAquick Gel Extraction Kit (28706) were from Qiagen. Erastin (S7242) was from Selleckchem. 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, 238813), deferoxamine mesylate (D9533), ferrostatin-1 (SML0583), iron(III) nitrate (F8508) were from Sigma-Aldrich; KI696 (HY-101140) from MedChemExpress shRNAs were obtained from the The RNAi Consortium (TRC) or identical sequences cloned into pLKO.1p: shRFP, TRCN0000072203; shGFP, TRCN0000072186; shNFS1, TRCN0000229753; shTFRC, TRCN0000057659; shIRP1 TRCN0000056553; shFXN, TRCN0000006138 and TRCN0000006137; and shISCU, TRCN0000290051 and TRCN0000289988.
Rneasy plus mini kit
Manufactured by Takara Bio
Sourced in Japan
The RNeasy Plus Mini Kit is a product from Takara Bio designed to facilitate the extraction and purification of total RNA from various sample types. It utilizes a silica-membrane-based method to efficiently capture and purify RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt master mix, by Takara Bio (2 mentions)
Hcc1954, by American Type Culture Collection (1 mentions)
Hcc70, by American Type Culture Collection (1 mentions)
Erastin s7242, by Selleck Chemicals (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Hs578t, by American Type Culture Collection (1 mentions)
Bt549, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Efm19, by Leibniz Institute DSMZ (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantitect rev transcription kit, by Qiagen (1 mentions)
Reverse transcriptase m mlv rnase h, by Takara Bio (1 mentions)
Kod dna polymerase, by Takara Bio (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Paraformaldehyde solution, by Solarbio (1 mentions)
Dimethyl sulfoxide (dmso), by MP Biomedicals (1 mentions)
9 protocols using rneasy plus mini kit
1
Comprehensive Reagents and Cell Lines
2
Quantification of Lineage-Specific Transcripts
3
RNA Extraction and Sequencing of PAMs
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Total RNA was extracted from primary PAMs and iPAMs using an RNeasy Plus Mini kit (Takara). Reverse Transcriptase M-MLV (RNase H-) (TaKaRa) was used to make cDNA, which was then amplified by KOD DNA polymerase (Takara) using primers (F: 5′-GTAATAATACAAGAAGATT-3′ and R: 5′-TCATTGTACTTCAGAGTGGTC-3′). Gel electrophoresis was used to analyze the obtained PCR products, which were then purified with a Gel Extraction Kit (OMEGA) according to the manufacturer’s instructions. The purified PCR products were further analyzed by DNA sequencing.
4
Quantifying Neuroinflammatory Markers in Ischemic Brain
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Total RNA was isolated from primary microglia cells or mouse brain tissue in the fringe area of the infarct using the RNeasy Plus Mini Kit (TaKaRa), according to the manufacturer’s instructions. After reverse transcription, quantitative real-time PCR was performed using primers specific for the genes encoding TREM2 and the inflammatory mediators IL-1β, IL-10, inducible nitric oxide synthase (iNOS), and TNF-α. Fast thermal cycling was performed using a real-time PCR system (Roche LightCycler 480) under the following conditions: pre-denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15s, 57°C for 30s, and 72°C for 30s. Semi-quantitative PCR experiments were also performed using primers that specifically amplified the C-terminal region of the full-length transcript encoding TREM2. The results were expressed as the relative mRNA expression of the threshold cycle value, and were normalized by parallel amplification of the endogenous control GAPDH. The relative mRNA expression level in the control group (target mRNA/GAPDH value) was set to 100%, and the mRNA values in other groups were converted to fold changes after comparison with the control group.
5
Chondrocyte Culture and Characterization
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The ssDNA strands (Table 1 ) that were designed with our sequences were synthesized by Takara (Otsu, Japan). Fetal bovine serum (FBS), penicillin–streptomycin solution, phosphate-buffered saline (PBS), 0.25% (w/v) trypsin-ethylenediaminetetraacetic acid solution, type II collagenase, and Dulbecco’s modified Eagle medium (DMEM) were obtained from GE Healthcare (Little Chalfont, UK). Dimethyl sulfoxide (DMSO) was purchased from MP Biomedicals (California, USA). Wogonin was obtained from Coolaber (China, Beijing). Tris-HCl, MgCl2, bicinchoninic acid (BCA), and sodium dodecyl sulfate (SDS) were acquired from Bio-Rad (Hercules, CA). The culture vessels and culture plates were procured from Corning (NY, USA). Polyvinylidene difluoride (PVDF) membranes were acquired from Millipore (MA, USA). Antibodies (COL-II and AGC) were purchased from Abcam (Cambridge, UK). Phalloidin and DAPI were obtained from Cytoskeleton (Denver, USA). The 4% paraformaldehyde solution was acquired from Solarbio (Beijing, China). The SYBR® Green I polymerase chain reaction (PCR) master mix, RNeasy® Plus Mini Kit, and DNase I were obtained from Takara (Tokyo, Japan).
6
RNA-seq Data Analysis Pipeline
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Total RNA was extracted using the RNeasy Plus mini kit (740990.250; Takara), and its concentration was measured using NanoDrop (2000/2000c Spectrophotometers, Thermo Fisher Scientific). The Illumina software package, “bcl2fastq”, was used for base-calling. The raw reads were mapped to the human reference genome sequence GRCh38 using TopHat (ver. 2.1.1) combined with Bowtie2 (ver. 2.3.4.1). Differential expression analysis was performed using the edgeR package, the enriched analysis was based on Gene Ontology (GO) database, and the pathway analysis by using Parametric Gene Set Enrichment Analysis (PGSEA) package (Ge et al., 2018 (link), 2020 (link)).
7
Gene Expression Analysis of Cardiac Markers
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Total RNA was extracted using the RNeasy Plus mini kit (740990.250; Takara) and cDNA was synthesized using the SuperScript VILO cDNA synthesis kit (11754-250; Thermo Fisher Scientific). Next, qRT-PCR was performed using the Taqman Fast advanced master mix (4444557; Thermo Fisher Scientific) and assessed using the ViiA 7 real-time PCR system (Thermo Fisher Scientific). The expression of the target gene was normalized to that of 18S rDNA, the internal control (18s rRNA, Hs99999901_s1). The genes analyzed using the TaqMan gene expression assays were as follows: MYH6 (Hs01101425_m1), MYH7 (Hs01110632_m1), MYL2 (Hs00166405_m1), MYL7 (Hs01085598_g1), SCN5A (Hs00165693_m1), GJA1 (Hs00748445_s1), ATP2A2 (Hs00544877_m1), CACNA1C (Hs00167681_m1), KCNJ2 (Hs01876357_s1), KCNQ1 (Hs00923522_m1), and KCNH2 (Hs04234270_g1).
8
Quantitative RT-PCR Analysis of Gene Expression in Mouse Retina and Microglia
9
Quantitative RT-PCR Analysis of SMARCB1 and Neural Genes
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RNA was extracted from cell pellets using Qiagen RNeasy Plus minikit and converted to cDNA using Takara RNA to cDNA EcoDry premix. Reactions were run in triplicate using cDNA converted from 10 ng of RNA. Primers used for real-time PCR analysis of SMARCB1 and neural developmental genes are included in Supplemental Table S1 . Data were normalized to GAPDH expression.
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