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Noti hf1/21/2024 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). To avoid these, follow the recommended guidelines for storage and reactions, and always check for the efficacy of digestion along with purification of digested products on an agarose gel.GEO help: Mouse over screen elements for information. Star activity (or off-target cleavage) and incomplete cleavage are potential challenges which may occur due to suboptimal enzymatic conditions or inappropriate enzyme storage. For complete digestion, make sure that the enzyme volume is 1/10th of the total reaction volume, the optimal temperature is constantly maintained throughout the reaction, the total reaction time is appropriately calculated based on the amount of DNA to be digested, appropriate buffers should be used to ensure maximal enzymatic activity, and in case of a double digest, make sure that the two restriction sites are far enough so that the activity of one enzyme cannot interfere with the activity of the other. Always use a control DNA digestion with the enzyme to ensure adequate activity (to avoid interference due to high glycerol in the enzyme). The enzyme should always be stored at -20C and multiple freeze-thaw cycles should be avoided in order to maintain optimal activity. The most common challenges with restriction digest include- 1. The four most common types of restriction enzymes include: Type I (cleaves at sites remote from a recognition site), Type II (cleaves within or at short specific distances from a recognition site), Type III (cleave at sites a short distance from a recognition site), and Type IV (targets modified DNA- methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA). Restriction Enzymes NotI A restriction enzyme or restriction endonuclease is defined as a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at or near that site, known as restriction site or target sequence.
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