4200 tapestation d1000 screentape

Ligated and purified libraries were amplified using KAPA HiFi HotStart Real-time PCR 2X Master Mix (KAPA Biosystems). AT libraries were amplified with 2 μL of KAPA P5 primer and 2 μL of SureSelect P7 Index primer. SS libraries were amplified with 5 μL of SWIFT-1S P5 Index and P7 Index primers. BE samples were amplified with 5 μL of KAPA P5 and KAPA P7 primers. The reactions were denatured for 45 s (s) at 98 °C and amplified 13–15 cycles for 15 s at 98 °C, for 30 s at 65 °C, and for 30 s at 72 °C, followed by final extension for 1 min at 72 °C. Samples were amplified until they reached Fluorescent Standard 3, cycles being dependent on input DNA quantity and quality. PCR reactions were then purified using 1 × AMPure XP bead clean-up and eluted into 20 μL of nuclease-free water. The resulting libraries were analyzed using the Agilent 4200 Tapestation (D1000 ScreenTape) and quantified by fluorescence (Qubit dsDNA HS assay). Primers used in the study are summarized in Additional file 1: Table S2.

The sequencing libraries were prepared from total RNA (700 ng) using the TruSeq Stranded Total RNA Kit (Illumina, Inc), following the manufacturer's instructions. Adapter‐ligated libraries were amplified by PCR, purified using AMPure XP beads, and validated for appropriate size on a 4200 TapeStation D1000 Screentape (Agilent Technologies, Inc). The DNA libraries were quantitated using a KAPA Biosystems qPCR Kit and were pooled together in an equimolar fashion. The pooled libraries were then denatured and diluted to 16 pM for On‐Board Cluster Generation and sequencing on a HiSeq 2500 sequencer using the appropriate single‐read cluster kit and rapid mode SBS reagents for 50‐cycle single‐read sequencing following the manufacturer's recommended protocol (Illumina, Inc.). The RNA sequencing data from this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under the accession number GSE154832 (http://www.ncbi.nlm.nih.gov/geo/).

Amplified cDNA was generated using the SMART-Seq v4 Ultra Low Input RNA kit (Clontech, Mountain View, CA). Total RNA (10 ng) was used to synthesize first-strand cDNA utilizing proprietary template switching oligos. Amplified double stranded cDNA is created by LD PCR using blocked PCR primers and unique sample barcodes were incorporated. The resulting cDNA was purified using AmpureXP beads (Beckman Coulter, Brea, CA). Abundant Ribosomal cDNA was then depleted using R probes and 12 cycles of PCR with universal PCR primers completed the library. The final libraries were purified using AmpureXP beads, and validated for appropriate size on a 4200 TapeStation D1000 Screentape (Agilent Technologies,, Santa Clara, CA). The DNA libraries were quantitated using KAPA Biosystems qPCR kit and pooled together in an equimolar fashion. Each pool was denatured and diluted to 16pM for On-Board Cluster Generation and sequencing on a HiSeq2500 sequencer for 100 paired-end sequencing following the manufacturer’s recommended protocol (Illumina Inc., San Diego, CA).