1/4/2024 0 Comments Noti hf![]() ![]() For applications that require product analysis by fluorescence excitation (e.g. Note: The FastDigest Green Buffer offers the same high performance in DNA digestion and downstream applications as the colorless FastDigest Buffer. Restriction fragment length polymorphism (RFLP).100% buffer compatibility with downstream applications.100% activity of all FastDigest enzymes in the universal buffer.Short incubation times and optimal composition of the universal FastDigest Buffer eliminate star activity effects. Therefore, enzymes for downstream applications can be directly added to the FastDigest reaction mix.įor additional convenience FastDigest Green Buffer includes a density reagent and two tracking dyes for direct loading of digestion reaction products on gels. DNA modifying enzymes, such as Klenow Fragment, T4 DNA Ligase, alkaline phosphatases and T4 DNA Polymerase all have 100% activity in FastDigest Buffer. See Reaction Conditions for FastDigest Enzymes for a table of enzyme activity, heat inactivation and incubation times for this and other FastDigest restriction enzymes. The universal buffer allows rapid single-, double-, or multiple DNA digestion within 5–15 minutes eliminating any need for buffer change or subsequent DNA clean-up steps. Thermo Scientific FastDigest NotI is one of an advanced line of fast restriction enzymes that are all 100% active in the universal FastDigest and FastDigest Green reaction buffers. GSM7091149_Balbc_da圓_macrophage_rep2_rpkm.Thermo Scientific FastDigest NotI restriction enzyme recognizes GC^GGCCGC site and cuts best at 37☌ in 5–15 minutes using universal FastDigest Buffer. Skeletal muscle regeneration failure in ischemic-damaged limbs is associated with pro-inflammatory macrophages and premature differentiation of satellite cells Pro-inflammatory macrophages impair skeletal muscle regeneration in ischemic-damaged limbs by inducing precocious differentiation of satellite cells Supplementary files format and content: indexed binary format files include RPKM values for each sample The bigwig files were generated using bamCoverage from alignment of reads (parameter-normalizeUsing:RPKM ). The reads from fastq files were aligned to GRCm38 reference genome using STAR v2.7.4a (parameter-sjdbOverhang: 99) DNA fragments between 400-600bp were selected by gel extraction using Zymoclean Gel DNA recovery kit (Zymo, #D4002). Library PCR was performed using unique combinations of Nextera-PCR i5/i7 primers. A Zymo DNA clean and concentrator kit (Zymo, R1014 ) was utilized (PMID: 24385147). DNA was digested by NotI-HF (NEB, #R3189L), tagmented using Tn5 assembled with adaptors Tn5ME-A/Tn5MErev and Tn5ME-B/Tn5MErev. PCR preamplification of cDNA was performed with IS PCR and Tn5ME-A-aHic using 2X KAPA PCR mix (Kapa Biosystem, KK2602) followed by cleanup using SPRISelect beads (Beckman Coulter, REF B23319). Total RNA was extracted using TRIzol Reagent (Invitrogen) according to manufacturer’s protocol.įirst-strand reverse transcription and template switching was performed using an Oligo(dT) primer (dT30VN-ME-A), a locked nucleic acid-containing TSO (NotI-TSO), and Superscript IV reverse transcriptase (Invitrogen, # 18090050). Approximately 100,000 macrophages for collected from one mouse for each biological replicate. Macrophages were isolated by fluorescence-activated cell sorting (SonySorter SH800s) with gating for PI-/CD45+/CD11b+/F480+ cells. Streptavidin-PE/Cy7 (Biolegend, 405206) was used as a secondary reagent for anti-F4/80-biotin (PMID: 25896247). Primary antibody staining was performed using anti-CD45-Alexa Fluor 488 (clone HI30, Invitrogen, MHCD4520), anti-CD11b (clone M1/70, Invitrogen, 12-0112-81), and anti-F4/80-biotin (clone A3-1, Bio-rad, MCA497BT) antibodies for 40 minutes. Cells were blocked with purified anti-mouse CD16/32 antibody (Biolegend, #101301) for 10 minutes. Single-cell suspensions were generated from skeletal muscle tissue using mechanical dissociation and enzymatic digestion with 0.05% Pronase (Sigma, 537088) for one hour. Macrophages from ischemic hindlimb skeletal muscle were isolated on post-op day 3 for RNA-seq analysis. ![]() Hindlimb ischemia surgery was performed on young adult male mice as previously described (PMID: 18285563). GEO help: Mouse over screen elements for information. ![]()
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