Truseq sr cluster kit v4 reagents

Total RNA was extracted from sorted cells using the RNeasy plus mini kit (Qiagen) according to the manufacturer's instructions. Clontech RNA-seq libraries were prepared for the splenic NK cell preparations. Double stranded cDNA for RNA-seq library preparation was generated using SMART-Seq v4 Ultra Low Input RNA reagents (Catalog Number 634888, Clontech) according to the protocol provided with the reagents beginning with 10 ng of total RNA and using 9 cycles of PCR. 150 pg of the resulting cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (Catalog Number 15032354, Illumina) using the single cell RNA-seq library preparation protocol developed for the Fluidigm C1 (Fluidigm).
TruSeq stranded mRNA-seq libraries were prepared for the bone marrow-derived NK cells. RNA-seq libraries were prepared using 400 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina) according to the protocol supplied with the reagents and starting with 100ng of total RNA on a Sciclone liquid handling robot (PerkinElmer) using a PerkinElmer-developed automated script.
Library sequencing cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500 using TruSeq SBS Kit v4 reagents. Sequencing data were processed using the Illumina Pipeline Software version 1.82.

Total RNA was extracted and DNAse-I treated using a spin column-based RNA purification kit (Macherey-Nagel). cDNA was prepared with SuperScript II reverse transcriptase (Invitrogen). Primers (listed in Supplemental Experimental Procedures) were used for SYBR Green qPCR (Applied Biosystems), and specificity was confirmed with dissociation curves. For mRNA sequencing, 100-bp single-end RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA reagents (Illumina). Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents. Sequencing was performed with Illumina HiSeq 2500 in 100-bp reads run. The RNA-seq reads were mapped to the mm9 genome using TopHat (Kim et al., 2013 (link)), allowing multimapped reads to be randomly assigned once among the mapped loci. Gene counts were generated with HTseq-count program using default parameters, and TE counts were computed using BEDtools (multicov). Sequencing depth normalization and differential expression analyses were performed using the voom function of Bioconductor package LIMMA (Law et al., 2014 (link)).