Vahts stranded mrna seq library prep kit

Mouse CRC paired tumor and colon tissues were isolated from AOM/DSS CRC bearing C57BL/6 mice as described above. Tissues were immediately frozen in liquid nitrogen after isolated and then stored at −80 °C refrigerator.
Total RNA from was isolated with RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and RNA integrity was determined using an Agilent Bioanalyzer4200 (Agilent technologies).
VAHTS stranded mRNA-seq Library Prep Kit (NR612, Vazyme) was used to prepare the sequencing library according to the manufacturer’s protocol. The cDNA library was sequenced using the Illumina sequencing platform (Novaseq). The RNA isolation, library construction and sequencing were performed at Shanghai Biochip Co., Ltd. For gene-expression analysis, the fragments of genes were counted and subsequently normalized by FPKM according to the following formula. $\mathrm{FPKM}=\frac{\mathrm{Total}\mathrm{exon}\mathrm{fragments}}{\mathrm{Mapped}\mathrm{reads}\left(\mathrm{millions}\right)\times \mathrm{exon}\mathrm{length}\left(\mathrm{KB}\right)}$

Total RNA was extracted by the TRIZOL (Ambion). The RNA was further purified with two phenol–chloroform treatments and then treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were redetermined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (Bio-Rad). The integrity of RNA was further verified by 1.5% agarose gel electrophoresis. For each sample, 1 μg of total RNA was used for RNA-seq library preparation by VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme). Polyadenylated mRNAs were purified and fragmented, and then converted into double strand cDNA. After the step of end repair and a tailing, the DNAs were ligated to VAHTS RNA Adapters (Vazyme). Purified ligation products corresponding to 200–500 bp were digested with heat-labile UDG, and the single strand cDNA was amplified, purified, quantified, and stored at -80℃.
For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina HiSeq X Ten system for 150 NT paired-end sequencing.

Total RNA was extracted with TRIZOL (Life Technology, Carlsbad, CA, USA) as previous study descripted [16 (link)]. The RNA was further purified with two phenol–chloroform treatments and then treated with RQ1 DNase (Promega, Madison, WI, USA) to remove DNA. The quality and quantity of the purified RNA were redetermined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (BioRad, Hercules, CA, USA). The integrity of RNA was further verified by 1.5% agarose gel electrophoresis. For each sample, 1 μg of the total RNA was used for RNA-seq library preparation by VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme, Nanjing, China). Polyadenylated mRNAs were purified and fragmented, and then converted into double strand cDNA. After the end repair and A tailing step, the DNAs were ligated to VAHTS RNA Adapters (Vazyme, Nanjing, China). Purified ligation products corresponding to 200‒500 bps were digested with heat-labile UDG, and the single strand cDNA was amplified, purified, quantified, and stored at − 80ºC before sequencing. For high-throughput sequencing, the libraries were prepared following the manufacturer’s instructions and applied to an Illumina HiSeq X Ten system for 150 nt paired-end sequencing.

Total RNA was extracted with TRIzol (15596-018, Ambion, United States) and purified twice with phenol–chloroform and RQ1 DNase treatment (M6101, Promega, Madison, WI, United States) to eliminate DNA. Absorbance at 260/280 nm (A260/A280) was read on a SmartSpec Plus instrument (Bio-Rad, United States) to quantify RNA and integrity assessed by 1.5% agarose gel electrophoresis.
Three biological replicates were prepared for TFRC-OE and NC samples. RNA-seq libraries were prepared from 1 μg total RNA using a VAHTS Stranded mRNA-seq Library Prep Kit (NR605-02, Vazyme, China), and polyadenylated mRNAs were purified, fragmented, and converted into double-stranded complementary DNAs (cDNAs). cDNAs were ligated to VAHTS RNA Adapters after end repair and A tailing, and products of 200–500 bps were produced during digestion with heat-labile uracil-DNA glycosylase (UDG). Single-stranded cDNAs were amplified, purified, quantified, and stored at −80°C. Then, 150 nt paired-end sequencing was performed using the Illumina NovaSeq 6000 system, following the manufacturer’s protocol.

Two biological replicates SERBP1-OE and control (Ctrl) cells were prepared for total RNA extraction. RNA was purified twice with phenol-chloroform treatment, and genomic DNA was removed with RQ1 DNase (Promega, Madison, WI, USA). The quality, quantity, and integrity of RNA were verified by absorbance at 260 nm/280 nm (A260/A280) (SmartSpec Plus; Bio-Rad, Hercules, CA, USA) and 1.5% agarose gel electrophoresis, respectively.
RNA-seq libraries were prepared by VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme, Nanjing, China) with one 1 microgram RNA as input, which was conducted in accordance with the published method (Li et al., 2018 (link)). We used Illumina HiSeq X Ten system for sequencing with 150 nt paired-end as parameter.

CAR-T was co-cultured with 1 × 10⁶ target cells at E: T = 1:1 for 48 h. CD3 + CAR-T cells were collected by flow cytometry. RNA was extracted and the concentration and purity were measured. OD260/OD280 within 1.8–2.1 was considered qualified. RNA sequencing was conducted by The Beijing Genomics Institute. PolyA RNA was purified from total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module. Library was constructed by VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme, China). Each group was sequenced in triplicate. The |log2(FoldChange)| ≥ 0.58 and P-value ≤ 0.05 genes were selected to be considered as the significant difference. The sequencing data have been deposited to the Sequence Read Archive (SRA) (PRJNA953945).
M5C sequencing was conducted by Shanghai Genechem Co.,Ltd. M5C-IP and library preparation were performed according to the published protocol [40 (link)]. Agilent 2200 system with HS RNA Reagent was used for RNA quality control. Purified RIP RNAs were reverse transcribed into cDNA sequencing library. Libraries were sequenced via Nova platform (Illumina). The sequencing data have been deposited to the SRA (PRJNA 957745).

Total RNA was extracted from RPL8-OE and Ctrl HeLa cells using TRIzol (Ambion). The purified RNAs were evaluated for purity and quantity using SmartSpec Plus (BioRad, USA). RNA-seq libraries were constructed using the captured polyadenylated RNAs and the VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme), as previously reported (Li et al., 2018 (link)). The RNA-seq libraries were then applied to 150-nucleotide (nt) paired-end sequencing using the Illumina HiSeq X Ten system.