Total RNA was isolated from biological triplicates of each cell line using the miRNeasy Mini RNA isolation kit (Qiagen) including a DNase I digestion step to remove genomic DNA. For all samples except K562 CYRANO+/+ and CYRANO−/− cells, library preparation was performed following a previously published protocol (43 (link)) with the following modifications. For each sample, 20 μg of total RNA was combined with 1 μL of 10 nM spike-in control oligos (Bioneer) before size fractionation on a 15% urea-polyacrylamide gel. RNAs were ligated to 3’ randomized adapter using T4 RNA ligase 2 truncated KQ (NEB) supplemented with 20% PEG 8000 (NEB) at 25°C overnight. The PCR-amplified cDNA was gel-purified using an 8% polyacrylamide gel. Library preparation and next generation sequencing for K562 CYRANO+/+ and CYRANO−/− cells was performed by DNA Link using the NEB Next Small RNA Library Prep kit for Illumina (New England Biolabs). Approximately 2 × 107 reads per sample were obtained and the first 18 nt of each read was mapped to mature miRNA sequences downloaded from miRbase (miRbase_v22) as described previously (10 (link), 44 (link)). EdgeR was used for differential expression analysis (45 (link)), and only miRNAs with a mean RPM > 1 in wild-type cells were used for subsequent analyses.
Mirneasy mini rna isolation kit
Manufactured by Qiagen
The MiRNeasy Mini Kit is a lab equipment product designed for the isolation of total RNA, including small RNAs such as miRNA, from various biological samples. It uses a silica-based membrane technology to efficiently capture and purify the RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Phusion polymerase, by Thermo Fisher Scientific (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Qiazol, by Qiagen (2 mentions)
Precellys evolution homogenizer, by Bertin Technologies (2 mentions)
Peg8000, by New England Biolabs (1 mentions)
Dna free kit, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Agilent bioanalyzer rna 6000 nano chip, by Agilent Technologies (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Dnase 1, by Qiagen (1 mentions)
5 protocols using mirneasy mini rna isolation kit
1
miRNA Sequencing from Total RNA
2
RNA Isolation and Small RNA Sequencing
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Tissues from E18.5 embryos were homogenized in Qiazol (Qiagen) using a Precellys Evolution homogenizer (Bertin Technologies). Total RNA was extracted using the miRNeasy Mini RNA isolation kit (Qiagen) and digested with DNase I to remove genomic DNA. Library preparation was performed as previously described (Kim et al. 2019 (link); Han et al. 2020 (link)). In brief, total RNA was size fractionated on a 15% urea-polyacrylamide gel. RNAs were ligated to a 3′ randomized adapter using T4 RNA ligase 2 truncated KQ in buffer supplemented with 20% PEG 8000 at 25°C overnight. Following gel-purification, RNAs were ligated to a 5′ randomized adapter using T4 ligase 1 in buffer supplemented with 20% PEG 8000 at 37°C for one hour. The product was then reverse transcribed with Superscript III (Invitrogen), cDNA was PCR amplified with Phusion polymerase (Thermo Fisher), and product was gel purified.
Northern blotting was performed as previously described (Han et al. 2020 (link)). In brief, total RNA was run on a 15% urea-polyacrylamide gel and transferred to a nylon membrane before UV-crosslinking, blocking, and probing with a radiolabeled probe. A locked nucleic acid probe (mmu-miR-450b-5p miRCURY LNA miRNA Detection probe; Qiagen) was used to detect miR-450b while standard DNA oligonucleotide probes were used for other miRNAs. Probe sequences provided inSupplemental Table S2 .
Northern blotting was performed as previously described (Han et al. 2020 (link)). In brief, total RNA was run on a 15% urea-polyacrylamide gel and transferred to a nylon membrane before UV-crosslinking, blocking, and probing with a radiolabeled probe. A locked nucleic acid probe (mmu-miR-450b-5p miRCURY LNA miRNA Detection probe; Qiagen) was used to detect miR-450b while standard DNA oligonucleotide probes were used for other miRNAs. Probe sequences provided in
3
RNA Extraction and cDNA Synthesis
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RNA was isolated using the miRNeasy Mini RNA isolation kit (Qiagen). DNAse digestion was performed using the DNA-free Kit (Applied Biosystems). cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). cDNA was amplified by PCR using Taq DNA polymerase (NEB) and the resulting product was run on TAE agarose gels. Oligonucleotide primers and PCR conditions are reported in Supplementary Table S2. Images were captured using a ChemiDoc XRS+ System and processed using ImageLab software (Bio-Rad).
4
miRNA Expression Profiling in Mouse Embryos
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Tissues from E18.5 embryos were homogenized in Qiazol (Qiagen) using a Precellys Evolution homogenizer (Bertin Technologies). Total RNA was extracted using the miRNeasy mini RNA isolation kit (Qiagen) and digested with DNase I to remove genomic DNA. Library preparation was performed as previously described (Kim et al. 2019 (link); Han et al. 2020 (link)). In brief, total RNA was size-fractionated on a 15% urea–polyacrylamide gel. RNAs were ligated to a 3′ randomized adapter using T4 RNA ligase 2 truncated KQ in buffer supplemented with 20% PEG 8000 overnight at 25°C. Following gel purification, RNAs were ligated to a 5′ randomized adapter using T4 ligase 1 in buffer supplemented with 20% PEG 8000 for 1 h at 37°C. The product was then reverse-transcribed with SuperScript III (Invitrogen), cDNA was PCR-amplified with Phusion polymerase (Thermo Fisher), and the product was gel-purified.
Northern blotting was performed as previously described (Han et al. 2020 (link)). In brief, total RNA was run on a 15% urea–polyacrylamide gel and transferred to a nylon membrane before UV cross-linking, blocking, and probing with a radiolabeled probe. A locked nucleic acid probe (mmu-miR-450b-5p miRCURY LNA miRNA fetection probe; Qiagen) was used to detect miR-450b, while standard DNA oligonucleotide probes were used for other miRNAs. Probe sequences are inSupplemental Table S3 .
Northern blotting was performed as previously described (Han et al. 2020 (link)). In brief, total RNA was run on a 15% urea–polyacrylamide gel and transferred to a nylon membrane before UV cross-linking, blocking, and probing with a radiolabeled probe. A locked nucleic acid probe (mmu-miR-450b-5p miRCURY LNA miRNA fetection probe; Qiagen) was used to detect miR-450b, while standard DNA oligonucleotide probes were used for other miRNAs. Probe sequences are in
5
RNA Isolation from Muscle Biopsies
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For RNA isolations, a small piece of each muscle biopsy sample (~ 10 mg) was submerged into 700 μl Qiazol lysis reagent (Qiagen cat.nr 79306) directly from − 80 °C. Tissue was lysed using an ultra-turrax T25 homogenizer and RNA was isolated using the miRNeasy mini RNA isolation kit (Qiagen cat.nr 217004), according to manufacturer’s protocol (including a 30 min on-column DNAse I (Qiagen cat.nr 79254) treatment). RNA quality was checked on an Agilent BioAnalyzer RNA Nano 6000 chip (cat.nr 5067–1511) or Agilent Fragment Analyzer and all RNA samples had an RNA Integrity Number (RIN)/RNA Quality Number (RQN) ≥ 6.6 (with 53/65 samples ≥ 8). Technical replicate samples of two FSHD-affected biopsies (FSHD-02_VL and FSHD-13_VL) were included in the two major sequencing batches to exclude any bias in signature detection. For this, a new sample of the identical muscle biopsy was included in the RNA isolation, sequencing library preparation and sequencing of the second batch of control biopsies.
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