Labchip rna pico sensitivity assay

Total RNA was isolated from green fluorescent protein (GFP)-positive, sorted cells using the RNeasy Mini Kit (Qiagen). RNA quality was checked using 2100 Bioanalyzer RNA 6000 Nanoassay (Agilent) or LabChip RNA Pico Sensitivity assay (PerkinElmer) before library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit (Illumina). Libraries were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies) Kapa Library Quantification kit (Kapa Biosystems) or low pass sequencing on a MiSeq Nano v2 run (Illumina). One hundred cycle paired end sequencing was performed on an Illumina HiSeq 2500, HiSeq 4000, or NovaSeq 6000. RNA isolation, library preparation, and sequencing were performed on three biological replicates. RNA-seq data were mapped as described previously^{18 (link)} and HTSeq^{30 (link)} (version 0.6.1p1) were used to get gene-level count and estimated FPKM based on GENCODE (vM9)^{34 (link)}. Voom^{35 (link)} was used for gene differential expression analysis after trimmed mean normalization.

For RNA-seq, RNA quality was checked by 2100 Bioanalyzer RNA 6000 Nano assay (Agilent) or LabChip RNA Pico Sensitivity assay (Perkin Elmer) before library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit (Illumina). ChIP-seq libraries were prepared from 2–10ng of DNA using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina with NEBNext Q5 Hot Start HiFi PCR Master Mix according to the manufacturer’s instructions (New England Biolabs) with the following modifications: a second 1:1 Ampure cleanup was added after adaptor ligation. The Ampure size selection step prior to PCR was eliminated. Completed ChIP-seq libraries were analyzed for insert size distribution on a 2100 BioAnalyzer High Sensitivity kit (Agilent) or Caliper LabChip GX DNA High Sensitivity Reagent Kit (Perkin Elmer). All libraries were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies), Kapa Library Quantification kit (Kapa Biosystems) or low pass sequencing on a MiSeq Nano v2 run (Illumina). One hundred cycle paired end sequencing (RNA-seq) or fifty cycle single end sequencing (ChIP-seq) was performed on an Illumina HiSeq 2500 or HiSeq 4000.

All library preparation and sequencing was carried out by the Hartwell Center at St Jude Children’s Research Hospital. For RNA-seq, RNA quality was checked by 2100 Bioanalyzer RNA 6000 Nano assay (Agilent) or LabChip RNA Pico Sensitivity assay (Perkin Elmer) before library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit (Illumina). For ChIP-seq, libraries were prepared from 5–10 ng of DNA using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina with NEBNext Q5 Hot Start HiFi PCR Master Mix according to the manufacturer’s instructions (New England Biolabs) with the following modifications: a second 1:1 Ampure cleanup was added after adaptor ligation. The Ampure size selection step prior to PCR was eliminated. Completed ChIP-seq libraries were analyzed for insert size distribution on a 2100 BioAnalyzer High Sensitivity kit (Agilent) or Caliper LabChip GX DNA High Sensitivity Reagent Kit (Perkin Elmer). All libraries were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies), Kapa Library Quantification kit (Kapa Biosystems) or low pass sequencing on a MiSeq Nano v2 run (Illumina). One hundred cycle paired end sequencing (RNA-seq) or fifty cycle single end sequencing (ChIP-seq) was performed on an Illumina HiSeq 2500 or HiSeq 4000.