The 16S rRNA V3–V4 library was constructed by two rounds of PCR with the following primers: 341F:5′TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGAGGCAGCAGCCTACGGGNBGCASCAG3′ and 805R:5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGACTACNVGGGTATCTAATCC3′ via reaction procedure (95 °C for 2 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min). PCR products were purified with 1 × KAPA AMPure beads (KAPA, Cat#KK8002). Then, products were put through a second PCR reaction procedure (95 °C for 2 min, followed by 8 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min). PCR products were purified with 1 × KAPA AMPure beads and analyzed using a Bioanalyzer DNA Kit, followed by quantification with real-time PCR. DNA libraries were pooled and sequenced on Illumina MiSeq (Illumina; CA) using a 2 × 250 bp paired-end protocol with overlapping reads.
Kapa ampure beads
Manufactured by Roche
KAPA AMPure beads are paramagnetic beads used for the purification and size selection of nucleic acids, such as DNA and RNA, during various molecular biology workflows. The beads selectively bind to nucleic acids based on their size and can be easily separated from the reaction mixture using a magnetic stand. This allows for the efficient removal of unwanted contaminants, salts, and primers, ensuring high-quality nucleic acid samples for downstream applications.
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2 protocols using kapa ampure beads
1
16S rRNA V3–V4 Amplicon Sequencing
2
RNA Extraction and Library Preparation for Sequencing
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For cultured MuSCs, RNA was extracted using TRIzol reagent (Simgen, 5301100). The RNA quality and concentration were determined using NanoDrop 2000 (Thermo, USA). For PFA fixed cells in vivo, RNA from 30k cells was extracted using a QIAGEN RNeasy FFPE Kit (Qiagen, 73504) according to the manufacturer’s instructions. RNA-seq library for fixed cells were constructed according to the protocol published previously [77 (link)]. In brief, 2.3 μL RNA was mixed with 1 μL 10 μM oligodT and 1 μL 10 mM dNTP, and then incubated at 72 °C for 3 min. RNA solution was mixed with RT reaction mixture (SuperScript II reverse transcriptase 100 U, RNAse inhibitor 10 U, Superscript II first-strand buffer 2 μL. DTT 5 mM, Betaine 1 M, MgCl2 6 mM, TSO 1 μM, Nuclease-free water). The library PCR product was purified with KAPA Ampure beads (KAPA, KK8001). A total of 50 ng of PCR product was tagmented and enriched using TruPre DNA Library Prep Kit (Vazyme, TD501-01). First-strand and tagmented DNA library size distribution were checked by Agilent 2100 bioanalyzer. For all samples, cDNA library with peak size of 1.5-2.0 kb and tagmented DNA with a broad peak size of 300-800 bp were accepted.
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