Rna cleanup kit

Mouse anti-fluorescein antibodies (anti-FAM) were purchased from Bialexa (Moscow, Russia). Oligonucleotides with modifications (5-carboxyfluorescein (FAM), biotin, 5-carboxyrhodamine-X [ROX], and BHQ2) were synthesized by Syntol (Moscow, Russia). DNAseI, EnGene LbCas12a, T7 RNA polymerase, RNAse inhibitor, NTP, Bst 2.0 WarmStart polymerase, an RNA cleanup kit, and a Monarch DNA gel extraction kit were purchased from NEB (Ipswich, MA, USA). Unmodified oligonucleotides, dNTP, Tersus polymerase, 100+ bp (100–1500 bp) DNA ladder, and M2 means 1 kb (250–10,000 bp) DNA ladder were obtained from Evrogen (Moscow, Russia). Protein A was produced by Imtek (Moscow, Russia). HAuCl₄ and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The membranes as components of lateral flow strips were purchased from Advanced Microdevices (Ambala Cantt, India). All salts and organic compounds had analytical-grade purity.

The RNA library for Direct RNA sequencing (SQK-RNA002) was prepared following the ONT direct RNA sequencing protocol version DRCE_9080_v2_revH_14Aug2019. Briefly, 500 ng poly-A RNA or poly-A tailed IVT RNA was ligated to the ONT RT adaptor (RTA) using T4 DNA Ligase (NEB, M0202M). Then the product was reverse transcribed using SuperScript^TM III Reverse transcriptase (Invitrogen, 18080044). The product was purified using 1.8× Agencourt RNAClean XP beads, washed with 70% ethanol and eluted in nuclease-free water. Then the RNA: DNA hybrid was ligated to RNA adapter (RMX) and purified with 1× Agencourt RNAClean XP beads and washed twice with wash buffer (WSB) and finally eluted in elution buffer (ELB). The FLO-MIN106D was primed according to the manufacturer’s protocol. The eluate was mixed with an RNA running buffer and loaded to the flow cell. MinKnow (19.12.5) was used to perform sequencing. Three replicates from different passages and different flow cells were used for each biological replicate. For Direct rRNA library preparation, total RNA was poly-A tailed using E. coli Poly(A) Polymerase (NEB, M0276) and purified using RNA Cleanup kit (NEB, T2040S) as in the above protocol.

Purified total RNA samples were treated with DNase I (NEB) with reaction buffer for 1h to remove the remaining plasmid and genome DNA. After digestion of the DNA, 0.5 M EDTA solution (Invitrogen™) were added to samples (1:100 dilution) to prevent Mg²⁺ dependent RNA hydrolysis. DNase I was denatured by heating at 75°C for 10 minutes. The RNA samples were further purified using an RNA-clean up kit (NEB). The concentration and quality of the purified total RNA samples were quantified via the 260/280 and 260/230 ratios using a Nanodrop 8000 spectrophotometer (Thermo Fisher).

DNA PCR primers corresponding to the 5' and 3' ends of the HIF-1α transcript were obtained (IDT, Coralville, Iowa). The 5’ primers contained a 20-base T7 RNA polymerase promoter sequence. Using a leukocyte cDNA library purchased from TaKaRa Corporation (TaKaRa Bio, San Jose, CA), PCR reactions were performed using an Avantage2 Taq polymerase APCR kit from TaKaRa. Various cycles were performed and the PCR products were monitored for expected bands and the absence of contaminating products of the wrong size by visualizing on an agarose gel. PCR products were cloned using a TA TOPO cloning kit (ThermoFisher, Waltham, MA). Individual clone DNAs were isolated (Qiagen, Baltimore, MD), digested with restriction enzymes, and run on agarose gels. Clones displaying different restriction enzyme digestion patterns were identified, and the plasmid inserts were Sanger sequenced at the JHU Sequencing Core Facility. This sequencing identified a predominant HIF-1α form (P1) and three transcriptional variants (V1, V2, and V3). The HiScribe T7 ARCA mRNA kit (NEB, Ipswich, MA) yielded full-length transcripts with a 5’-methyl cap and a 3’ poly-A tail. Each mRNA reaction was purified by an RNA Cleanup kit (NEB, Ipswich, MA), quantitated, and visualized on an agarose gel.

Following IVT, 30 µL of nuclease-free water was added to each IVT reaction product to increase the reaction volume. Each IVT product was then purified using a Monarch® RNA Cleanup Kit (NEB). 100 µL of RNA binding buffer was added to each 50 µL IVT product followed by the addition of 150 µL of absolute ethanol. Each mixture was then transferred to a provided silica-based spin column and centrifuged in a table-top centrifuge. 500 µL of ethanol-based DNA wash buffer was added to each spin column and centrifuged as above. The washing step was repeated once more and the flow-through from all steps was discarded. Each spin column was transferred to a clean microcentrifuge tube and 10 µL of the provided nuclease-free water was added directly to the matrix of each spin column followed by centrifugation for DNA collection. All centrifugation steps were carried out in 16,000 rcf for 60 s.

RNA was extracted using Trizol and cleaned up with the Monarch Total RNA Miniprep Kit or RNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Equal amounts of RNA extracted from three biological replicates were pooled for RNA-sequencing to mitigate batch effects.