Single read cluster generation kit v4

Using 1 μg of total RNA, cDNA libraries were prepared using the mRNA-seq 8-Sample Prep Kit as per manufacturer’s instructions (Illumina, RS-100-0801). Briefly, poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, mRNA was fragmented to ~200 base pair fragments and cDNA generated with ligated adaptors. These products were purified and PCR enriched to create the final cDNA library. Single-Read Cluster Generation Kit v4 was used for cluster generation as per manufacturer’s instructions (Illumina, GD-103-4001). Briefly, cDNA library samples were bound to complementary adapter oligonucleotides grafted on the surface of the Illumina Genome Analyzer flow cell. The templates were copied from the hybridized primer by 3′ extension using a high fidelity DNA polymerase. The 36 Cycle Sequencing Kit v4 on the Genome Analyzer II X platform was used for sequencing (FC-104-4002). The phi X 174 (PhiX) bacteriophage genome DNA was used as a control lane to validate sequencing quality. Samples were sequenced to 42 bp. After 40 bp the sequencing quality at 3′ ends significantly decreased under the acceptable threshold of 20 and sequencing reads were therefore trimmed to 40 bp to ensure acceptable quality. RNA-seq data sets were uploaded to GEO with a series accession number GSE52652.

RNA was isolated from C57BL/6 and Stat2^−/− mouse lungs using the Agilent RNA miniprep kit. RNA integrity was determined using an Agilent 2100 bio analyzer. mRNA was purified by using Sera-Mag Oligo(dT) Beads, fragmented with magnesium-catalyzed hydrolysis, and reverse transcribed into cDNA using random primers (Superscript II; Invitrogen). Then, cDNA underwent end repair with T4 DNA polymerase and Klenow DNA polymerase, followed by the addition of “A” bases to the 3′ end and ligation to adaptor oligonucleotides. Products from the ligation were run on a 2% agarose gel. A gel slice consisting of the 200 bp region (±25 bp) was excised and used as a template for PCR amplification. The final PCR product was purified, denatured with 2 N NaOH, and diluted to 10–12 pM prior to cluster amplification on a single-read flow cell v4, as outlined in the Single-Read Cluster Generation Kit v4 (Illumina). The flow cell was sequenced on an Illumina Genome Analyzer II. The data were analyzed as previously described (24 (link)). Full sequencing data has been uploaded to the National Center for Biotechnology Information Gene Expression Omnibus, GSE119029.

DNA fragments (150 to 250 bp) were selected for library construction and sequencing libraries were prepared using the ChIP-Seq Sample Preparation Kit (Illumina; San Diego, California, USA; Cat. No. IP-102-1001) according to the protocol supplied with the reagents. Prior and post library construction, ChIP products were quantified using a Qubit fluorometer (Invitrogen; Carlsbad, California, USA). One lane of each library was sequenced on the Illumina Genome Analyzer IIx using the Single-Read Cluster Generation Kit v4 and 36 Cycle Sequencing Kit v4. Data were processed using the Illumina Pipeline Software v1.60.