Agilent 2100 expert software

The total RNA of CECs from 10 patients with FECD was isolated by the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol, as described in our previous report^{15 (link)}. Briefly, CECs lysed with QIAzol lysis reagent were thawed at 37 °C, mixed with140 μL chloroform, and centrifuged at 12,000 g at 4 °C for 15 min. The supernatant was collected and mixed with an equal volume of 70% ethanol, followed by concentration using spin columns. The quantity and quality of total RNA were determined using an Agilent 2100 Bioanalyzer with an RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA). The quality of total RNA was assessed by determining the RNA integrity number (RIN) using the Agilent 2100 Expert Software (Agilent Technologies).

Total RNA was isolated from 30 mg of grinded in liquid nitrogen and homogenized endometrial tissue with the use of a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and genomic DNA contamination was removed by DNAse treatment (RNase free DNAse Kit, Qiagen, Valencia, CA, USA). The initial RNA quality and quantity were determined spectrophotometry using NanoDrop ND-1000 (Thermo Scientific, Pittsburgh, PA, USA). Subsequently, RNA integrity was evaluated with microfluidic electrophoresis by 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA integrity number (RIN) was calculated for each sample using Agilent 2100 Expert software (Agilent Technologies, Inc., Santa Clara, CA, USA). Only samples with a RIN above 8.0 were processed further.

The stability of fresh PH_(1-110)GFP NPs and dehydrated PH_(1-110)GFP NPs stored for two years at room temperature (R.T.D.) was compared. The PH_(1-110)GFP NPs were dehydrated using the vacufuge™ concentrator 5301 (Eppendorf, Germany) at a centrifugal force of 240 g at 30°C for 30 min and stored at room temperature (28-30°C) for two years. The Agilent Bioanalyzer 2100 (Agilent Technologies, USA) equipped with the Protein 230 assay kit was used to evaluate the degradation of PH_(1-110)GFP NPs R.T.D. Electrophoretic assays were performed following the manufacturer’s instructions (Supplementary Figure 2). Finally, the results were analyzed with Agilent 2100 expert software (Agilent Technologies, USA).

RNAs were extracted from the samples using the NucleoSpin RNA Kit (Macherey Nagel, UK), following a protocol for small RNA purification. Extracted RNA samples were analyzed on an Agilent 2100 Bioanalyzer using the agilent 2100 Expert Software in conjunction with the Bio‐SmallChip, which is especially indicated for small RNAs (length < 200 nucleotides). RNA integrity and quantification were assessed by performing a smear analysis of the electropherograms obtained by the capillary electrophoresis.

Viable cells were counted with Trypan Blue (Thermo Fischer Scientific, Cat#T8154) solution, and for each cell line, 1,000–2,000 viable cells were loaded into a chip and processed with the Chromium Single Cell Controller (10X Genomics, Pleasanton, California United States, estimated recovery: 1,500 cells/sample). Single-cell RNA-Seq libraries were prepared using the Single Cell 3′ GEM, Library and Gel Bead Kit V3 (10× Genomics, Cat#PN-1000075) according to manufacturer instructions. Briefly, single cells were partitioned into individual Gel Beads-in-emulsion (GEMs) and the RNA obtained from lysed cells was barcoded through reverse transcription. Each cell is encapsulated in a gel bead that contains a unique 14- base pair (bp) molecular barcode, a 10-bp randomer to index molecules (unique molecular identifier, UMI), and an anchored 30-bp oligo-dT to prime polyadenylated RNA transcripts. DynaBeads® MyOne Silane Beads (Thermo Fischer Scientific, Cat#37002D) were used to purify the resulting barcoded cDNA that was subsequently amplified via PCR (12-14 cycles, according to the available cDNA quantity). Libraries were then checked and quantified via the Agilent 2100 Expert Software. The resulting libraries were sequenced across one lane using an Illumina Hiseq4000 machine (Supplementary Table S12).

The concentration (OD260) and purity (OD260/280 ratio) of extracted total RNA was measured using NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, MA, USA). Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to assess the RNA integrity using the Eukaryote total RNA 6000 Pico LabChip kit and the Eukaryote total RNA Pico assay according to the manufacturer’s instructions. The RNA integrity numbers (RIN) were calculated using the Agilent 2100 Expert Software (Agilent Technologies); RIN = 1 indicates low RNA quality and RIN = 10 indicates highest RNA quality.

RNA was isolated from flash frozen tissue using an RNeasy Kit (Qiagen), and 100 ng of RNA was processed by the Molecular Biology Core at Dana-Farber Cancer Center, Boston, MA, USA. Samples were assessed for quality and concentration (Agilent Bioanalyzer RNA Nano or Pico chips); smear analyzed (Agilent 2100 Expert software) to quantify the percentage of RNA fragments greater than 300 nt; capture and reporter Code sets added; samples hybridize at 65°C for 16 hours, washed and loaded onto a custom-made cartridge (XT-GXA-P1CS-096) using the nCounter Analysis System Prep Station. The cartridge was scanned using the nCounter Digital Analyzer at the maximum resolution of 1150 FOV. Data analysis was completed using nSolver software. Gene expression normalization was performed using the housekeeper genes, tbp and tubb5.