Clontech smarter ultra low input rna kit v4

ASPCs were sort-purified from visceral adipose tissue (epididymal WAT) of wild type C57BL/6 mice. Sorted cells were used to prepare RNAseq libraries by the Epigenomics Core at Weill Cornell Medicine using the Clontech SMARTer® Ultra® Low Input RNA Kit V4 (Clontech Laboratories). Sequencing was performed on an Illumina HiSeq 2500, yielding 50 bp single end reads. Raw sequencing reads were demultiplexed with Illumina’s CASAVA (v1.8.2). Adapters were trimmed from reads using FLEXBAR (v2.4) (41 (link)) and reads were aligned to the NCBI GRCm38/mm10 mouse genome using the STAR aligner (v2.3.0) (42 (link)) with default settings. Reads per gene were counted using Rsubread (43 ). Normalized counts per gene were calculated using using DESeq2 version 1.18.1 (44 (link)).

ILC2 and ILC3 were sort-purified from small intestine of Rorc(γt)-Gfp^TG mice or BMAL1^fl/fl and BMAL1^ΔRorc littermates. Sorted cells were used to prepare RNA-seq libraries by the Epigenomics Core at Weill Cornell Medicine using the Clontech SMARTer Ultra Low Input RNA Kit V4 (Clontech Laboratories). Sequencing was performed on an Illumina HiSeq 2500, yielding 50 bp single-end reads.
Raw sequencing reads were demultiplexed with Illumina CASAVA (v1.8.2). Adapters were trimmed from reads using FLEXBAR (v2.4) and reads were aligned to the NCBI GRCm38/mm10 mouse genome using the STAR aligner (v2.5.2b) with default settings. Reads per gene were counted using Rsubread. Genes with at least 50 or more counts in at least 2 samples were tested for differential expression. Differential expression was assessed using DESeq2 version 1.22.2 with default parameters and with a false discovery rate (FDR) of 0.1. Principal component analysis (PCA) was performed after applying the DESeq2 varianceStabilizingTransformation function, using the 500 genes with highest variance.

RNA-seq libraries were prepared from 1,000 sorted colonic lamina propria ILC2s (CD45⁺Lin⁻CD90.2⁺CD127⁺KLRG1⁺) by the Epigenomics Core at WCM using the Clontech SMARTer Ultra Low Input RNA Kit V4 (Clontech Laboratories). Libraries were sequenced on the Illumina HiSeq 2500 system, generating 50 bp single-end reads. Two samples from two control-diet-fed WT SPF mice and two samples from two inulin-fibre-diet-fed WT SPF mice were used. Reads were demultiplexed using Illumina’s CASAVA (v.1.8.2), adapter-trimmed using FLEXBAR (v.2.4)^{62 (link)} and aligned to the mouse genome (NCBI GRCm38/mm10) using STAR aligner (v.2.3.0)^{63 (link)} with the default parameters. Read counts per gene (RefSeq annotation) were determined using the Rsubread R package (v.3.10 I)^{64 (link)}. Normalization and differential expression analysis were performed using DESeq2 (v.1.26.0)^{65 (link)}. Before differential expression testing, genes were prefiltered to include only genes that had a minimum of 50 raw reads in at least two samples. Tests were corrected for multiple comparisons, using a FDR of 10% to determine significance. GO enrichment analysis was performed using the enrichGO function of the clusterProfiler R package (v.3.14.3)^{66 (link)}, using the biological process ontology. Enrichment P values were corrected for multiple comparisons using the Benjamini and Hochberg method⁶⁷ .