Agilent high sensitivity rna screentape

Total RNA from each sample was extracted using an RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). The quality of the extracted RNA was estimated with an Agilent High Sensitivity RNA ScreenTape using TapeStation 2200 (Agilent Technologies, Santa Clara, CA, USA). PolyA RNA isolation was performed with a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA). A NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) and a NEBNext Multiplex Oligos for Illumina were used for the preparation of libraries. The preparation of the libraries was performed in biological triplicate for Panc1/siNeg, Panc1/siSOX9, Colo357/siNeg, and Colo357/siSOX9. The sequencing was performed with an Illumina NovaSeq 6000 System (Illumina, San Diego, CA, USA). The quality of raw reads was assessed using the MultiQC tool. The raw reads were mapped on the human reference genome (hg38) using HISAT2 (Galaxy Version 2.1.0 + galaxy 5, usegalaxy.org, accessed on 25 May 2022). The featureCounts (Galaxy Version 1.6.4 + galaxy 1, usegalaxy.org, accessed on 25 May 2022) was used for counting reads to GENCODE release 33 annotated genes [24 (link)]. The obtained read counts were converted into transcripts per kilobase million (TPM) values [25 (link)].

Molecular analyses were performed on a total of 28 patients. Biopsy samples were disrupted and homogenized in presence of Tungsten Carbide Beads, 3 mm diameter (QIAGEN, Hilden, Germany) with the TissueLyser II (QIAGEN). RNA was extracted from the homogenized samples using the Maxwell^® RSC miRNA Tissue Kit, with the automated Maxwell instrument, according to the manufacturer’s protocol. RNA was quantified by Qubit fluorometer with Qubit™ RNA HS Assay Kit (Thermo Fisher Scientific) and RNA quality was assessed with the Agilent High Sensitivity RNA Screen Tape (Agilent Technologies) on an Agilent-4200 Tapestation, obtaining a mean RNA integrity number (RIN) value of 7.05 (max value 10, min value 3.7).

Cells were collected before TF induction (day 2 of RA/SAG differentiation) and 48 h after Dox treatment (day 4 of RA/SAG differentiation). RNA was extracted by using TRIzol LS Reagent (Life Technologies) and purified using the RNAeasy Mini Kit (Qiagen). Agilent High Sensitivity RNA Screentape (Agilent, 5067-5579) was used to check RNA integrity. A 500 ng quantity of RNA was used to prepare RNA-seq libraries and spiked-in with ERCC ExFold Spike-In mixes (Thermo Fisher Scientific, 4456739). RNA-seq libraries were prepared using a TruSeq Stranded mRNA Library Prep kit (Illumina, 20020594). Library size was verified using High Sensitivity DNA ScreenTape (Agilent, 5067-5584). The KAPA Library Amplification kit was used on a Roche LightCycler 480 for library quantification before pooling. The libraries were sequenced on an Illumina NextSeq 500 using V2.5 chemistry (75 cycles, single-end 75 bp) at the Genomics Core Facility at New York University.

Total RNA from endometrial biopsies was isolated using QIAsymphony RNA kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. RNA concentrations were quantified using a Multiskan GO spectrophotometer (Thermo Fisher Scientific, Waltham, USA) at a wavelength of 260 nm. Integrity of the total RNA samples was evaluated by the RNA integrity number (RIN) and DV200 metrics using an Agilent high-sensitivity RNA ScreenTape in a 4200 TapeStation system (Agilent Technologies Inc, Santa Clara, CA). Samples used for the global RNA-seq showed RIN values ranging from 4.9 to 9.2.

A complete list of primers used for qPCR can be found in Supplementary Table 1. Total RNA was extracted from dissected retinas using Trizol (10296010; Invitrogen, Grand Island, NY) using standard manufacturer’s recommendations. RNA quality of RIN 7 was confirmed using Agilent High Sensitivity RNA screen Tape. cDNA was synthesized using 500 ng of total RNA per biological sample using iScript Advanced cDNA Synthesis (1725037; Bio-Rad, Hercules, CA). Quantitative PCR was performed on 1 µl of cDNA per biological sample using iTaq Universal SYBR Green Supermix (1725121, Bio-Rad) and a Bio-Rad CFX Real-Time System. Mouse Tctn1 exon2 was targeted using primers spanning the Tctn1 exon 2/exon 3 junction. To determine Tctn1 mRNA levels we generated a standard curve of known concentrations of full-length mouse Tctn1 plasmid (10 ng/µl–0.0001 pg/µl) and approximate Tctn1 mRNA levels were calculated by plotting the averaged Cq values from each biological samples to the standard curve.