title: GSM8824137: Spleen, O. tsutsugamushi Gilliam-infected C57BL/6 mice, Day8, replicate 1, RNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Female C57BL/6J mice (8-12 weeks-old, purchased from the Jackson Laboratory #000664) were intravenously inoculated with 6.8 × 104 FFU, 200 µl of Karp or Gilliam strain, or PBS (mock). Mice were sacrificed at established timepoints, and spleens were collected in RPMI 1640 medium. Spleens were then stored in RNALater. Total RNA was extracted by using the RNeasy Mini kit (Qiagen). Total RNA (≥ 1ug) was sent to Novogene (Sacramento, CA) for Poly(A) RNA-Seq Sequencing Service. Provided by Novogene: Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dTTP for non strand specific library or dUTP for strand specific library. For the non strand specific library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the strand specific library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.  The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.