Rna clean and concentrator

Constructs were snap frozen in liquid nitrogen and stored at -80°C. RNA was extracted using Trizol in a Cryo-Cup grinder (BioSpec products, Bartlesville, UK) on dry ice. Total RNA was prepared with the Direct-Zol RNA miniprep kit and RNA clean and concentrator (Zymo Research, Cambridge, UK) according to the manufacturer’s instructions. Libraries were prepared using the NEBNext Poly(A) mRNA magnetic isolation module (New England Biosystems, Ipswich, UK) and then the NEBNext ultra-directional RNA library prep kit was used to create the final library. The sample was sequenced using 41bp paired end configuration with an Illumina NextSeq 500 sequencing device. Reads were trimmed using trimmomatic (20 (link)), pseaudoaligned using kallisto (21 (link)) with an index built from the hg38 cDNA fasta reference sequence, and then quality of the pseudoalignment was assessed using FastQC. Differential gene expression was performed using the sleuth package. Genes were considered differentially regulated based on an adjusted p value < 0.01. Differentially expressed genes between two groups (constructs at 0 weeks and after 8 weeks of treatment with osteogenic medium) were analyzed for enriched terms using GSEA in R. Heatmaps were generated in R using ggplot2. GO and KEGG (GSEA) analysis was performed on WebGestalt.

For characterizing the effect of freeze thaw on cell free mRNA expressions, RNA was extracted using plasma processed with S1, S2, S1FR, S2FR, and S1FRS2 conditions. Cell free mRNA was isolated by using plasma/serum circulating and exosomal RNA purification Kit (Norgen Biotek, cat. 42800) followed by 10X Baseline-ZERO DNase treatment (Epicentre, cat. DB0715K). DNase treated RNA samples were purified and further concentrated using RNA clean and concentrator (Zymo Research, cat. R1014). The purified RNA samples were assayed by RT-qPCR using custom selected 16 primers targeting MTND2, PPBP, B2M, PF4, ACTB, CORO1C, GSE1, GAPDH, SMC4, HBG1, NUSAP1, MIKI67, FGB, APOE, FGG, and ALB. Template RNA was mixed with Superscript III One-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen, cat. 11-732-020) to generate cDNA according to the protocol. PCR amplification products were treated with Exonuclease I (New England Biolabs, cat. M0293L) to digest single stranded primers at 37 °C for 30 min followed by inactivation of enzymes at 80 °C for 15 min. For RT-qPCR, cDNA from preamplification was diluted 1:80 and set-up in 96-well plates with SsoFast EvaGreen supermix with low ROX (BioRad, cat. 1725211) with above primers at 10 µM. QuantStudio 7 Flex (Applied Biosystems) was used to run RT-qPCR assay according to manufacturer’s recommended cycling conditions.

Total RNA was extracted from tissues using TRIzol (Invitrogen) according to the manufacturer’s instruction. For T2D and mouse liver samples, mRNA was further purified with Dynabeads mRNA DIRECT purification kit (Thermo Fisher, cat. 61,011). mRNA was adjusted to 15 ng/μl in 100 μl and fragmented using Bioruptor ultrasonicator (Diagenode) with 30 s on/off for 30 cycles. m⁶A-immunoprecipitation (m⁶A-IP) was performed using EpiMark N6-Methyladenosine enrichment kit (NEB cat. E1610S). RNA eluted from m⁶A-IP was cleaned using RNA Clean and Concentrator (Zymo Research, cat. R1013). Input and IP samples were then used to prepare library with KAPA mRNA Hyper Kit (Roche, Cat. KK8541). For fallopian tube and omental tumor tissues, total RNA was fragmented and directly subjected to m⁶A-IP. Takara Pico-Input Strand-Specific Total RNA-seq for Illumina (Takara, Cat. 634413) was used to construct libraries from total RNA where ribosome-derived cDNA was removed before final library amplification. T2D and mouse liver libraries were sequenced by the HiSeq4000 platform at SE50 mode. The ovarian cancer libraries were sequenced by the NextSeq 500 platform at PE37 mode.

RNA was isolated with Trizol (TRI REAGENT Sigma), DNAse-treated, and cleaned over a column (RNA Clean and Concentrator ZYMO Research). First-strand cDNA was synthesized in duplicate from each sample (Invitogen SuperScript III). SYBR green was used to perform qRT-PCR (ABI 7900). For each primer set, a standard curve from genomic DNA accompanied the duplicate cDNA samples (Glover-Cutter et al., 2008 (link)). mRNA levels relative to N2 control were determined by normalizing to the number of worms and the geometric mean of three reference genes (cdc-42, pmp-3, and Y45F10D.4; [Hoogewijs et al., 2008 (link)]). At least two biological replicates were examined for each sample. For statistical analysis, one sample t-test, two-tailed, hypothetical mean of 1 was used for comparison using Prism 6.0 software (GraphPad).

Escherichia coli strain Rosetta 2 DE3 carrying plasmid pMJ806 was obtained from Addgene. The Cas9 protein was prepared following the protocol as described (Anders and Jinek, 2014). For in vitro transcription of guide RNA, pT7‐sgRNA derived gRNA‐specific construct was linearized at a unique restriction enzyme site NcoI, followed by dephosphorylation with Shrimp Alkaline Phosphatase (rSAP) (New England Biolabs, Ipswich, MA), and then purified with DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA). sgRNA was synthesized by using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) following the manufacturer's instructions and purified with RNA Clean and Concentrator (Zymo Research, Irvine, CA). For each DNA/ribonucleoprotein reaction, 1 μg of sgRNA and 1 μg of Cas9 protein were mixed in Cas9 reaction buffer and incubated at room temperature for 15 min to form a ribonucleoprotein (RNP) complex. The resulting RNP was used for 2–3 reactions each with about 1 μg of PCR product in a total volume of 10 μL at 37 °C for 3 h. After inactivated at 65 °C for 15 min, the reactions were analysed with electrophoresis in 2% agarose gel.