Nebnext rrna depletion kit v2 human mouse rat

Frozen tissue (~ 1 g) was homogenized in 2 mL of Trizol (Thermo Fisher) using 2.8 mm ceramic beads (Hard Tissue Homogenizing Mix, VWR). RNA was extracted with a modified Trizol method as follows: after the addition of chloroform and phase separation of the Trizol lysate, the aqueous phase was combined with an equal volume of 100% ethanol and loaded onto a Zymo-Spin column and purified using the Quick-RNA Prep Kit (Zymo Research). For all samples, RNA concentration was measured with a Nanodrop (Thermo Fisher), and integrity was determined with a Fragment Analyzer (Agilent). If high molecular weight material was evident in the Fragment Analyzer trace, indicating the presence of genomic DNA, samples were treated with DNAse following the instructions of the Zymo RNA RNA Clean & Concentrator Kit (Zymo Research). Ribosomal RNA was depleted with the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat; New England Biolabs) using 500 ng input total RNA. All RNA-seq libraries were generated with the NEBNext Ultra II Directional library prep kit (New England Biolabs) and 2 × 150 nt paired-end reads were generated on a NovaSeq6000 instrument (Illumina).

rRNA depletion was performed on the extracted total RNAs from tissue and subsequent library prep following the NEBNext^® rRNA Depletion Kit v2 (Human/Mouse/Rat) (Cat#7400L, New England BioLabs, England)’s protocol, which depletes both mitochondrial (12S and 16S) and cytoplasmic (5S, 5.8S, 18S, and 28S) rRNA species. cDNA synthesis was performed by using Maxima First Strand cDNA Synthesis Kit for RT-qPCR, with dsDNase (Cat#1671, Thermo Scientific™, USA). End-repair, A tailing, adaptor ligation, and library amplification were performed by using the KAPA HyperPlus kit (Cat#KK8512, Rocha, USA). Completed libraries were quantified by each library by Qubit™ 1X dsDNA High Sensitivity (HS) assay kit (Cat#Q33231, Invitrogen™, USA) and the insert size estimation was measured by TapeStation, using D1000 ScreenTape assay (Cat#5067-5582, Agilent, USA).

mRNA samples were prepared in three biological replicates. Cells were grown in 15 cm dishes to 70–80% confluency. Cell pellets were resuspended in 1.5 mL TRI reagent (Sigma). 300 µL Chloroform (Applichem) was added, and samples were mixed and centrifuged at 4 °C, max. speed for 15 min. The upper phase was transferred to a fresh tube and subjected to isopropanol precipitation. Totally, 120 µg of RNA were treated with 100 U DNase I (Roche) at 37 °C for 30 min and purified by phenol-Chloroform extraction and Ethanol precipitation. 100 µg total RNA was used as an input for mRNA enrichment. Polyadenylated RNA was isolated using Dynabeads mRNA Purification Kit (Invitrogen) according to the manufacturer’s instructions. rRNA was depleted using NEBNext rRNA depletion kit v2 (Human/Mouse/Rat) (New England Biolabs) according to the manufacturer’s instructions, followed by another round of mRNA enrichment using Dynabeads mRNA Purification Kit (Invitrogen). The absence of rRNA in the final mRNA preparations was confirmed by capillary electrophoresis on a Fragment Analyzer (Supplementary Fig. 14).