Clinical samples for analytical validation and comparison with orthogonal approaches were received as whole blood or archival frozen plasma stored at −80°C. For blood samples, 16 to 20 mL peripheral blood was collected in Cell Free DNA Blood Collection Tubes (Roche, Pleasanton, CA) or Cell-Free DNA BCT tubes (Streck Inc., La Vista, NE). To isolate plasma: i) whole blood was centrifuged at 1600 × g for 20 minutes at room temperature, ii) supernatant was collected and centrifuged at 16,000 × g for 20 minutes at 4°C, and iii) supernatant was collected as plasma that underwent cfDNA extraction. Plasma was treated with proteinase K for 20 minutes at 60°C and mixed with 1.25 × volume of cfDNA binding solution (Thermo Fisher Scientific, Waltham, MA) and 500 ng/mL of paramagnetic MyOne SILANE beads (Thermo Fisher Scientific). Beads were washed twice with cfDNA wash solution (Thermo Fisher Scientific) and twice with 80% ethanol, and they were eluted in cfDNA elution solution (Thermo Fisher Scientific). cfDNA concentration was determined using the D1000 ScreenTape assay on the 4200 TapeStation (Agilent Technologies, Santa Clara, CA). cfDNA (20 to 100 ng) was used for library construction.
Myone silane beads
Manufactured by Thermo Fisher Scientific
MyOne SILANE beads are magnetic particles coated with a silica layer. They are designed for use in various applications that require the capture, separation, and purification of target molecules from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Tapestation 4200, by Agilent Technologies (1 mentions)
Cell free dna blood collection tubes, by Roche (1 mentions)
D1000 screentape assay, by Agilent Technologies (1 mentions)
Qubit rna br assay, by Thermo Fisher Scientific (1 mentions)
Epimark n6 methyladenosine enrichment kit, by New England Biolabs (1 mentions)
Zymo rna clean and concentrator 5, by Zymo Research (1 mentions)
T4 rna ligase 1, by New England Biolabs (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
3 protocols using myone silane beads
1
Plasma cfDNA Isolation and Quantification
2
M6A RIP Enrichment and Quantification
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M6A RIP was performed using EpiMark® N6-methyladenosine Enrichment Kit (NEB) following the manufacturer’s instructions with some modifications. Purified RNA was sheared to <350 bp using a Covaris ME220 and then purified using Zymo RNA Clean and Concentrator 5 (Zymo) following the manufacturer’s instructions. Purified RNA was then measured for concentration using the Qubit BR RNA Assay (ThermoFisher Scientific) following the manufacturer’s instructions, and 1 µg was used for each pulldown with 2 µL of M6A antibody. Following 1 h incubation and washes, RNA was eluted and purified with MyOne Silane Beads (ThermoFisher Scientific) then prepared for reverse transcriptase in which 1 µL from pulldown or input was put in reverse transcriptase reaction. Resulting cDNA was then used for qPCR using primers designed to predict M6A sites located on BDNF-AS (Supplementary Table 2 ). M6A sites were predicted using SRAMP37 (link). The data were analyzed using the ∆∆Ct method, normalizing to input, and the data are expressed as fold change relative to controls.
3
Isolation and Sequencing of 5' UTRs
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