Smarter ultra low input rna kit for sequencing v4

Total RNA was processed for library construction by Cofactor Genomics (St. Louis, MO, USA) according to the following procedure. Briefly, total RNA was reverse-transcribed using an Oligo(dT) primer, and limited cDNA amplification was performed using the SMARTer Ultra Low Input RNA Kit for Sequencing—v4 (Takara Bio, Shiga, Japan). The resulting full-length cDNA was fragmented and tagged, followed by limited PCR enrichment to generate the final cDNA sequencing library (Nextera® XT DNA Library Prep, Illumina, San Diego, CA, USA). Libraries were sequenced as single-end 75 base pair reads on the Illumina NextSeq500 per the manufacturer’s instructions.

The cDNA libraries were prepared using the SMARTer Ultra Low Input RNA Kit for Sequencing—v4 (TAKARA Bio) and Nextera XT DNA Library Prep Kit (Illumina) as per manufacturer’s instructions. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). The libraries were pooled and diluted to 3 nM using 10 mM Tris-HCl, pH 8.5 and then denatured using the Illumina protocol. The denatured libraries were loaded onto an S1 flow cell on an Illumina NovaSeq 6000 (Illumina) and run for 2 × 50 cycles according to the manufacturer’s instructions. De-multiplexed sequencing reads were generated using Illumina bcl2fastq (released version 2.18.0.12) allowing no mismatches in the index read.

Total RNA was extracted and processed for library construction by Cofactor Genomics (St. Louis, MO) according to the following procedure: briefly, total RNA was extracted from cell pellets using the protocol for Arcturus PicoPure™ RNA Isolation Kit (Thermo Fisher, Waltham, MA). Total RNA was reverse-transcribed using an Oligo(dT) primer, and limited cDNA amplification was performed using the SMARTer® Ultra® Low Input RNA Kit for Sequencing—v4 (Takara Bio USA, Inc., Mountain View, CA). The resulting full-length cDNA was fragmented and tagged, followed by limited PCR enrichment to generate the final cDNA sequencing library (Nextera® XT DNA Library Prep, Illumina, San Diego, CA). Libraries were sequenced as single-end 75 base reads on a NextSeq500 (Illumina, San Diego, CA) following the manufacturer’s protocols.

RNA isolation and purification were performed using Direct-zolTM RNA MicroPrep according with manufacturer instructions (Zymo Research, Irvine, CA) on three individual oocytes. Briefly, frozen eggs were thawed at room temperature and resuspended in Tri Reagent, followed by addition of 95% ethanol, DNase I treatment, and centrifugation in an IC column [34 (link)]. Final RNA elution was performed using DNase/RNase-free water. Total RNA was processed for library construction by Cofactor Genomics (St. Louis, MO) according to the following procedure: Briefly, total RNA was reverse-transcribed using an Oligo (dT) primer, and limited cDNA amplification was performed using the SMARTer® Ultra® Low Input RNA Kit for Sequencing – v4 (Takara Bio USA, Inc., Mountain View, CA). The resulting full-length cDNA was fragmented and tagged, followed by limited PCR enrichment to generate the final cDNA sequencing library (Nextera® XT DNA Library Prep, Illumina, San Diego, CA). Libraries were sequenced as single-end 75 base pair reads on an Illumina NextSeq500 following the manufacturer’s instructions.

RNA from all samples was extracted in house and shipped to Cofactor Genomics (St. Louis, MO, USA) on dry ice for library preparation and sequencing. RNA quality was checked using a Bioanalyzer. Samples having good RIN numbers (>7) were selected for RNA-seq library preparation (mRNAble Cat No. CFG001-30) and subsequent sequencing steps were performed by Cofactor Genomics. Total RNA reverse transcription was performed with an Oligo(Dt) primer followed by limited cDNA amplification using the SMARTer^® Ultra^® Low Input RNA Kit for Sequencing–v4 (Takara Bio USA, Inc., Mountain View, CA, USA). Next, the full-length cDNA was subject to fragmentation followed by tagging. The final sequencing cDNA library was generated using limited PCR enrichment (Nextera^® XT DNA Library Prep, Illumina, San Diego, CA, USA). Single-end 75 base pair reads were generated using the Illumina NextSeq500 platform. Resulting FASTQ files containing all the reads were provided to us on a URL.