Caliper labchip gx

Illumina library preparation, hybridization capture, and sequencing was conducted at the Yale Center for Genomic Analysis (YCGA). Library preparation was conducted using a modified Roche/Nimblegen SeqCap EZ Library Short Read protocol [68 ]. Library concentration was determined using PicoGreen assay (Invitrogen) and size selection was performed on a Caliper LabChip GX instrument (PerkinElmer).

Total RNAs were isolated using Trizol reagent (Life Technologies), phenol-chloroform extraction and isopropanol precipitation. RNAs were analyzed using a microchip capillary electrophoresis system (Caliper LabChip GX, PerkinElmer, Waltham, MA, USA). RNA with RQS >7 (RNA Quality Score) were treated with ribonuclease-free Turbo-DNase (Life Technologies) before reverse transcription (RT). RTs were performed with Moloney murine leukemia virus reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. Real-time quantitative PCR was performed with a ViiA™ 7 sequence detection application program using the following labeled ready-to-use primers/probe mixes (Life Technologies): Notch1 (Hs01062014_m1), Notch2 (Hs01050702_m1), Notch3 (Hs01128541_m1), Notch4 (Hs00270200_m1), DLL1 (Hs00194509_m1), DLL3 (Hs00213561_m1), DLL4 (Hs00184092_m1), Jag1 (Hs01070036_m1), Jag2 (Hs00171432_m1), HES1 (Hs00172878_m1), HEY1 (Hs01114113_m1), IRF5 (Hs00158114_m1), STAT1 (Hs01013996_m1), SOCS3 (Hs02330328_s1), TNF (Hs01113624_g1), IL-6 (Hs00174131_m1), IL-1β (Hs0155410_m1), CD40 (Hs00334176_m1), c-myc (Hs00811069_g1). QPCR for β actin (Actb) (Hs99999903_m1) was used as an endogenous control and for data normalization. Relative gene expression was calculated according to the 2^−ΔΔCt method.

Small ear biopsies were taken from 10-day-old rats to perform genotyping as previously described (51 (link)). Briefly, samples were digested overnight at 56°C in 350 μL of lysis buffer, then diluted at 1:20 in ultrapure water and added to 24 μL of PCR mix reaction prepared according to the manufacturer protocol (Herculase II Fusion DNA Polymerase, Agilent Technologies). The following primers were used to carry out the PCR amplification: forward primer 5′-TCAAGAGTGCCCTGTTCTAG-3′, reverse primer 5′-CTGGGGTGGTGTCAGTAAG-3′. The amplification program was run on a Veriti Thermal Cycler (Applied Biosystems) and consisted of 1 cycle at 95°C for 5 minutes, 35 cycles at 98°C for 10 seconds, 60°C for 10 seconds, and 72°C for 30 seconds, followed by 1 cycle at 72°C for 4 minutes. Finally, to determine the presence of mutations in the Aire gene, the PCR product’s length was estimated using a heteroduplex mobility assay with a microfluidic capillary electrophoresis system (Caliper LabChip GX, PerkinElmer).

Prior to 10X Genomics lrWGS library construction, genomic DNA samples were checked for fragment size distribution and were quantified. Genomic DNA fragment size distributions were determined with a Caliper Lab Chip GX (Perkin Elmer) to quantify DNA above 40 kb in length. Size selection was performed on 1.2 ug of genomic DNA with an 0.75% Agarose cassette on the Blue Pippin platform (Sage Science) with target specifications set to start at 40 kb and end at 80 kb. Samples were quantified using the Quant-it Picogreen assay kit (Thermo Fisher) on a Qubit 2.0 Fluorometer (Thermo Fisher) and normalized to a starting concentration of 1 ng/uL with TE (0.1 mM EDTA). Starting concentrations of 1 ng/uL were confirmed by picogreen and libraries were subsequently created in accordance with the 10X WGX protocol (10X Genomics). Library size was determined using the DNA 1000 Kit and 2100 BioAnalyzer (Agilent Technologies) and quantified using quantitative PCR (qPCR) (KAPA Library Quantification Kit, Kapa Biosystems). The finished WGX libraries were run on an Illumina HiSeqX platform at paired 151 bp reads with an eight-base single index read at the Broad Institute. Upon completion of sequencing, the resulting BCL files were processed by the Long Ranger Pipeline (10X Genomics) for alignment, variant discovery, and phasing.

DNA libraries were prepared with Nextera XT DNA Library Preparation Kit (Illumina, California, USA). Quality control was performed with Caliper LabChip GX (Perkin Elmer) prior to shotgun sequencing with MiSeq (Illumina, California, USA), with an expected sequencing depth of 260 Mb/library (expected coverage > 80×). One hundred twenty-nine million reads were generated (704 thousand reads/sample s.d. 349 thousand).
Sequences were pre-processed by removing low-quality (mean quality lower than 25) or low-complexity reads, reads mapping to human genome or to large and small ribosomal units of bacteria, fungi and human, and known contaminants (e.g. phiX174, Illumina spike-in). All genomes are available at the NCBI Sequence Read Archive (BioProject accession number PRJNA400143).