Superscript 2 reverse transcriptase

Total RNA from lysed cells was extracted from the purified B cells with the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Reverse transcription was conducted using Superscript II Reverse Transcriptase (TAKARA Bio) with random hexamer primer and oligo‐dT. Real‐time RT‐PCR was performed using commercially available TaqMan gene expression probes (Applied Biosystems) for human B cell–related genes, including bach2, id3, cd69, xbp1, irf4, prdm1, ctla4, id3, tlr4, nfam1, peli1, zap70, stk39, nod2 and gapdh. The expression level of each gene of interest was normalized to GAPDH, and the results were given as relative copy numbers.
Gene expression in mouse B cells including TLR4, Myd88, Bach2, NFAM1 and CTLA4 was determined by real‐time RT‐PCR using SYBR‐Green qPCR Master Mixes (Thermo Fisher Scientific). The relative fold change was calculated based on the 2^−ΔΔCt method. The relative quantity of the target mRNA was normalized to the level of β‐actin mRNA.

Total RNA from PBMC of 30 RA patients and 24 healthy controls were extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Total RNA (1 μg) was used for cDNA synthesis with oligodT primers (Invitrogen, Karlsruhe, Germany) and superscript II reverse transcriptase (Takara Bio, Shiga, Japan). PCR was performed using PCR Master (Roche, Mannheim, Germany) with the following primers: for PD-1 mRNA, 5′-CTCAGGGTGACAGAGAGAAG-3′ (forward) and 5′-GACACCAACCACCAGGGTTT-3′ (reverse) and for GAPDH, 5′-GTGAAGGTCGGAGTCAACG-3′ (forward) and 5′-TGAGGTCAATGAAGGGG-TC-3′ (reverse). Fold changes were normalised based on GAPDH expression, and each assay was conducted in a 96-well ABI 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA). This procedure was performed in triplicate.

ADSCs were lysed with isogen reagent (Nippon Gene, Tokyo, Japan) for RNA extraction. Total RNAs were reverse transcribed with SuperScript II Reverse Transcriptase (Takara) into cDNA. The full length human RAGE CDS sequence was amplified by PCR and specific primers with kpn1 restriction sites were designed according to sequence number NM_001206954.1 on (http://www.ncbi.nlm.nih.gov ). Full length RAGE was inserted into a pCDNA3.1 vector to construct a mammalian expression plasmid (PCDNA3.1-RAGE). Successful insertion was confirmed by sequencing.

Single worm RNA sequencing used a protocol modified from single-cell RNA sequencing for mouse cells^{62 (link)}. A single young adult animal was transferred into 2 μl lysis buffer and lysed by grinding. In all, 1 μl of each oligo-dT primer (10 μM) and dNTP (10 mM) was added into the PCR tube and heated at 72°C for 3 mins then cooled for 2 min. 6 μl reverse transcription mixture (100 U SuperScript II reverse transcriptase (Takara), superscript II first-strand buffer, 1 U RNAase inhibitor (Vazyme),10 M betaine (Sigma), 6 mM MgCl₂ (Ambion), and 100 μM TSO primer) was then added directly and incubated using thermal cycle: 90 min at 42 °C, 15 min at 72 °C and hold at 4 °C. cDNA samples were amplified with 10 μl KAPA HiFi HotStart ReadyMix (Kapa Biosystems) and 12.5 μl 10 μM IS PCR primers. The purified cDNA was fragmented by TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Inc) Hiseq X 10 system sequencing. Primers are listed in Supplementary Table 3.

In addition to the three cell types mentioned above, we used samples from 51 patients with CRC. A total of 51 CRC and matched adjacent normal (distance to cancer greater than 5 cm) tissue samples used for RT-qPCR assay were obtained from patients who had been diagnosed with CRC by pathological examination of tissue biopsy and undergone operations at the Affiliated Hospital of Qingdao University. No radiotherapy or chemotherapy was applied before tissue collection. Informed consent was obtained from all of the participating patients. This work was approved by the Research Ethics Committee of The Affiliated Hospital of Qingdao University and was performed following the 1964 Helsinki Declaration and its later revisions. We used an RNeasy kit (Beyotime, Shanghai, China, R0027) following the manufacturer’s instructions to extract RNA from the cells and tissues. Then, we reverse-transcribed 1 μg of total RNA using SuperScript II reverse transcriptase (TaKaRa, Japan, RR047). Quantitative PCR analysis was performed using SYBR Green Master Mix (TaKaRa, Japan, RR820) with an ABI 7900 HT real-time PCR system. The primer sequences for RT-qPCR are listed in Supplementary Table S1.