Bioanalyzer high sensitivity dna assay

Whole transcriptome library preparation was performed using the SMARTer® Stranded Total RNA-Seq Kit v3 –Pico Input Mammalian (Takara Bio Europe, France) according to the manufacturer’s instructions. Briefly, 10 ng total RNA was fragmented, cDNA synthesis was performed using random N6 primers, multiplexing indices and adapters were added, and ribosomal cDNA depletion was performed. Due to the fragmented nature of the RNA extracted from FFPE tissue, the fragmentation step was omitted in the library preparation of RNA from FFPE tissues. AMPure XP beads (Beckman Coulter, USA) were used instead of the recommended NucleoMag NGS Clean-up and Size Select beads (Macherey-Nagel, Germany). The qualities of the cDNA libraries were assessed using the Bioanalyzer High Sensitivity DNA assay with the 2100 Bioanalyzer system (Agilent, USA). The quantities of the cDNA libraries were assessed using the 7500 Real-Time PCR System (Applied Biosystems, USA) with the KAPA Library Quantification Kits–Complete kit (ABI Prism) (Kapa Biosystems, USA). Paired-end sequencing (2 x 100 bp) was performed on a NovaSeq 6000 instrument using the NovaSeq 6000 S1 Reagent Kit v1.5 (Illumina, USA).

The Ribo-seq protocol was modified according to a previous study using 2.5 × 10⁶ cells or approximately 10 µg of RNA^{38 (link)}. After optimization, we lowered the input to 50 mouse oocytes. Fifty denuded mouse oocytes were lysed in Ribo-seq lysis buffer (1% Triton X-100, 20 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and 100 µg/µL cycloheximide). The total DNA and RNA were digested by a mix of 1 U TURBO™ DNase (AM2239, Invitrogen), 0.02 U RNase A, and 0.8 U RNase T1 Cocktail (AM2286, Invitrogen) at 37 °C for 30 min. The digestion was terminated using SUPERase•In™ RNAase inhibitor (AM2696, Invitrogen). RNAs were purified using TRIzol (15596026, Invitrogen) in phasemaker tubes (A33248, Invitrogen), and the resulting RNAs were treated with T4 PNK (M0201S, NEB) to conduct end-repair. Ribosome-protected fragments (RPFs) were separated using 40% RNA PAGE-gel. Resulting 26‒40 nt bands were cut and minced using Squisher-Single (H1001, ZYMO) and soaked in recovery buffer (300 mM NaOAc pH 5.5, 1 mM EDTA, 0.25% v/v SDS, 10 mM MgCl₂) overnight. The RPFs were recovered by isopropanol precipitation. Libraries were then constructed using a SMARTer® smRNA-Seq Kit for Illumina (635030, TAKARA). Finally, samples were quantified using the Bioanalyzer High-Sensitivity DNA assay (Agilent Technologies) and sequenced using the Novaseq 6000 platform.

As input, 500 ng of total DNA per sample were utilized for preparing targeted methylseq libraries with Illumina TruSeq Methyl Capture EPIC Library Preparation Kit (Illumina (San Diego, CA, USA)). All steps were performed as recommended in Illumina user document 1000000001643 v01 May 2017. Furthermore, one additional purification step was introduced at the end of the standard procedure, using 1x Agencourt® AMPure® XP Beads (#A63881; Beckman Coulter, Inc.). The KAPA Hifi HotStart Uracil+Ready Mix 2x enzyme is needed to amplify the enriched libraries but is not substitutable and not included with the kit. DNA libraries were indexed and amplified with 11–13 cycles of PCR. Fragment length distribution of individual libraries was monitored using Bioanalyzer High Sensitivity DNA Assay (5067-4626; Agilent Technologies (Santa Clara, CA, USA). Quantification of libraries was performed by use of the Qubit^® dsDNA HS Assay Kit (Q32854; ThermoFisher Scientific).

Cells were filtered through a 70 µm filter and further diluted to target 8000 cells per run. Single cells were then partitioned and barcoded with the Chromium Controller instrument (10X Genomics) and Single Cell 3’ Reagent (v3.1 chemistry) kit (10X Genomics) according to the manufacturer’s specifications with no modification (Rev C). Final libraries were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and diluted to 3 ng/µL in buffer EB (Qiagen, Hilden, Germany). Library quality and concentration was confirmed using the Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies, Santa Clara, CA) prior to sequencing.