Kapa stranded rna seq library prep kit

Total RNA was extracted from exosomes isolated from M0 and M2 macrophages using TRIzol reagent (Invitrogen, Carlsbad, CA, United States). We obtained approximately 3 μg total RNA from every specimen using the VAHTS Total RNA-seq (H/M/R) Library Prep Kit from Illumina (Vazyme Biotech, Nanjing, China) to eliminate ribosomal RNA, but retained other RNAs like ncRNA and mRNA. We treated the purified RNA with RNase R (40 U, for 3 h at 37°C) (Epicenter), which was followed by TRIzol purification. We constructed a RNA-seq library using the KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) and deep sequencing with a HiSeq 4000 (Illumina, San Diego, CA, United States) conducted by Aksomics (Shanghai, China).

The total RNA content in each sample ranged from 1 to 2 μg, and mRNA enrichment was performed using the NEB Next^® Poly(A) mRNA Magnetic Isolation Module kit (New England Biolabs Inc., Ipswich, MA, USA, Cat No. E7490S). Then the library was constructed using a KAPA Stranded RNA-Seq Library Prep Kit (Roche Inc., Boston, MA, USA, Cat No. KK8401). After denaturation with 0.1 M NaOH, the single-stranded DNA was diluted to 8 pM and then amplified in situ with a TruSeqSR Cluster Kit v3-cBot-HS (Illumina Inc., San Diego, CA, USA, Cat No. PE-401-3001). The end of the resulting fragment was sequenced for 150 cycles on an Illumina HiSeq4000 sequencer.

We extracted total RNA from two pairs of BC and adjacent noncancerous tissues or T24 cells with or without small interfering RNA targeting circRGNEF (si-circRGNEF) via TRIzol reagent (Invitrogen, Carlsbad, CA, USA). We subjected nearly 3 μg of total RNA from every sample to VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) to eliminate ribosomal RNA. We retained other RNA such as non-coding RNA and mRNA. We treated purified RNA with RNase R (Epicenter, 40 U, 37°C for 3 h), and then performed TRIzol purification. We prepared RNA-Seq libraries through the KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland), which were subjected to deep sequencing through Illumina HiSeq 4000 at Aksomics, Inc., Shanghai, China (accession number: H1712024).

Total RNA was extracted from three paired NSCLC tissues and adjacent noncancerous lung tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Approximately 3 μg of total RNA from each sample was subjected to the VAHTS Total RNA-seq (H/M/R) Library Prep Kit from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) to remove ribosomal RNA while retaining other types of RNA, including mRNA and ncRNA. Purified RNA was treated with RNase R (Epicenter, 40 U, 37 °C for 3 h), followed by purification with TRIzol. RNA-seq libraries were prepared using the KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) and subjected to deep sequencing with an Illumina HiSeq 4000 at Aksomics, Inc., Shanghai, China (Accession code: H1712024).

Total RNA from the sepsis model and sham-operated mouse liver tissues was extracted with TRIzol Reagent (Invitrogen, CA, USA). Further, ~3 μg total RNA from every sample was subjected to a VAHTS Total RNA-Seq (H/M/R) Library Prep Kit (Vazyme Biotech Co., Ltd, Nanjing, China) to eliminate ribosomal RNA and retain other classes of RNAs such as noncoding RNAs and mRNAs. We treated purified RNA employing 40 U RNase R (Epicenter, New England Biolabs, MA, USA) at 37°C for 3 h, followed by TRIzol purification. Our lab used a KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) to prepare an RNA-Seq library, which was subjected to deep sequencing with Illumina HiSeq 4000 (CA, USA) at Aksomics, Inc. (Shanghai, China).

We extracted total RNA from three paired NSCLC tissues as well as adjacent non-cancerous lung tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). We subjected 3 μg of total RNA from each sample to the VAHTS Total RNA-Seq (H/M/R) Library Prep Kit from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) to remove ribosomal RNA and different classes of RNA such as non-coding RNA and mRNA. We treated purified RNA with RNase R (Epicentre Technologies, Madison, WI, USA; 40 U, 37 °C, 3 h), followed by TRIzol purification. We prepared RNA-Seq libraries using the KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) and subjected them to deep sequencing with the Illumina HiSeq 4000 at Aksomics, Inc. (Shanghai, China; accession code: H1712024).
For miRNA and mRNA analysis, A549 cells transfected with siRNA against circ-EPB41 or a negative control (NC) vector were used for high-throughput RNA-Seq as previously described (accession code: H1712024).