title: GSM1210263: strain B6 Influenza infection at 11 hours, repeat 1; Mus musculus; RNA-Seq
library_construction_protocol: For RNA isolation, each organ was cut into small pieces in the presence of QIAzol, homogenized using SPEX CertiPrep homogenizer, and total RNA was extracted using miRNeasy Mini Kit (Qiagen). The RNA integrity number (RIN) was determined using the TapeStation System (Agilent Technologies). Quantity was determined by Qubit Fluorometric Quantitiation (Life Technologies). For preparation of RNA-seq libraries, total RNA was fragmented into average size of 300 nucleotides by chemical heat (95Oc) treatment for 4:30 minutes (NEBNext Magnesium RNA Fragmentation Module). The 3’ polyadenylated fragments were enriched by selection on poly dT beads (Dynabeads Invitrogen). Strand specific cDNA was synthesized using a poly T-VN oligo (18 T) and Affinity Script RT enzyme (Agilent). Double strand DNA was obtained using Second strand synthesis kit (NEB). DNA ends were repaired using T4 polynucleotide kinase and T4 polymerase (NEB-Next). After addition of an adenine base residue to the 5’ end using Klenow enzyme (NEB-Next), a barcode Illumina compatible adaptor (IDT) was ligated to each fragment. The washed DNA fragment was amplified by PCR (12 cycles) using specific primers (IDT) to the ligated adaptors. The quality of the library was analyzed by Tapestation (Agilent).
attributes.tag: GEO Accession
attributes.value: GSM1210263