title: Sample 8
organism: Homo sapiens
description: Protocols: CD45+Lin-HLADR+CD11c+ cDC and CD45+Lin-HLADR+CCR7+ eTAC were sorted from the tonsils of 3 donors directly into Trizol Reagent. mTEC were sorted as CD45-HLADR+CD80+EpCAM+ cells from 3 separate donors. Consent to collect donor age and gender information was not provided. Total RNA was extracted using TRIzol reagent according to the manufacturers protocol as follows. 0.2x volumes of chloroform was added to the homogenate and the tube(s) were shaken vigorously. The tube(s) were incubated for 2-3 minutes at room temperature and centrifuged for 1 hour (4120 RCF, 4 °C) . Approximately 70% of the upper aqueous phase was transferred to a clean 15 mL tube with the addition of 1 μl of glycogen (20μg/μL) and 0.5x volume of isopropanol was added. The tube(s) were incubated overnight at -20 °C and centrifuged for 30 minutes (4120 RCF, 4 °C). The supernatant was removed and the pellet was washed twice with 80% ethanol. The total RNA pellet was air-dried for 8 minutes and dissolved in an appropriate volume of nuclease free water and quantified using Nanodrop UV-VIS Spectrophotometer. Quality and quantity of the total RNA was assessed by the 2100 bioanalyzer using a Pico chip. Strand-specific libraries were generated using the TruSeq RNA access sample preparation kit according to the manufacturer's instructions. The FF RNA was fragmented, random primed and reverse transcribed using SuperScript II Reverse Transcriptase with the addition of Actinomycin D. Second strand synthesis was performed using Polymerase I and RNaseH with replacement of dTTP for dUTP. The generated cDNA fragments were 3' end adenylated and ligated to Illumina sequencing adapters and subsequently amplified by 15 cycles of PCR. The libraries were analyzed on a 2100 BioAnalyzer using a 7500 chip. Capture pools of 200 ng per sample were compiled based on library yield and unique adapter sequence, where after target enrichment of the exome is performed. The captured fragments were amplified by a 2nd amplification of 10 PCR cycles. The captured fragments were assessed on a 2100 BioAnalyzer, diluted and equimolar pooled into a 10 nM multiplex sequencing pool, containing 12 samples per pool.