Umi second strand synthesis modules

RNA sequencing (RNA-seq) libraries were prepared from the RNA of cortex sections (Table 1). Sequencing and library preparation were performed by the DNA technologies and Expression Analysis Core in the Genome Center of the University of California, Davis. RNA Integrity (RIN) scores were assessed for all samples, resulting in a mean of 6.4 ± 0.87 (range = 4.9–8.3). Gene expression profiling was carried out using a 3′-Tag-RNA-Seq protocol. Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) for multiplexed sequencing according to the recommendations of the manufacturer starting from 300 ng total RNA each. Both the unique dual index (UDI)-adapter and unique molecular identifier (UMI) Second Strand Synthesis modules were used (Lexogen). The fragment size distribution of the libraries was verified via microcapillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA). The libraries were quantified by fluorometry on a Qubit fluorometer (Life Technologies, Carlsbad, CA) and pooled in equimolar ratios. The library pool was quantified via quantitative PCR (qPCR) with a KAPA Library Quantification Kit (Kapa Biosystems/Roche, Basel, Switzerland) on a QuantStudio 5 system (Applied Biosystems, Foster City, CA). The libraries were sequenced on a HiSeq 4000 sequencer (Illumina, San Diego, CA) with single-end 100-bp reads.

Gene expression profiling of primary enterocyte RNA samples and total intestinal RNA samples were carried out using a 3′-tag-RNASeq protocol. Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) for multiplexed sequencing according to the recommendations of the manufacturer using the UDI-adapter and UMI Second-Strand Synthesis modules (Lexogen). High integrity total RNA samples were processed according to the QuantSeq default protocol. The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA). The libraries were quantified by fluorometry on a Qubit fluorometer (LifeTechnologies, Carlsbad, CA), and pooled in equimolar ratios. The library pool was Exonuclease VII (NEB, Ipswich, MA) treated, SPRI-bead purified with KapaPure beads (Kapa Biosystems/Roche, Basel, Switzerland), quantified via qPCR with a Kapa Library Quant kit (Kapa Biosystems) on a QuantStudio 5 RT-PCR system (Applied Biosystems, Foster City, CA). Up to 48 libraries were sequenced per lane on a HiSeq 4000 sequencer (Illumina, San Diego, CA) with single-end 100 bp reads.

Total RNA from control (n=3) and Emx1-NICD (n=5) sorted cells was extracted using the Total RNA Purification Plus Kit (Norgen Biotek Corp., 48300). Gene expression profiling was carried out using a 3'-Tag-RNA-Seq protocol. Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen) for multiplexed sequencing according to the recommendations of the manufacturer using both the UDI-adapter and UMI Second-Strand Synthesis modules (Lexogen). The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer). Barcoded miRNA-Seq libraries were prepared using the NEXTflex Small RNA Sequencing kit V3 (PerkinElmer) with sequence randomized adapters according to the recommendations of the manufacturer. The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a Bioanalyzer 2100 (Agilent). Both sets of libraries were quantified by fluorometry on a Qubit instrument (Life Technologies), and then pooled in equimolar ratios. The library pools were quantified by qPCR with a Kapa Library Quant kit (Kapa Biosystems/Roche). Finally, both library sets were sequenced on a HiSeq 4000 sequencer (Illumina) with single-end 100 bp reads.