title: GSM6929055: Splenic B cells, O. tsutsugamushi-infected C57BL/6 mice, Day8, replicate 2, 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 a lethal dose (8 × 10e4 FFU, 200 µl) of bacteria or PBS (mock). Mice were sacrificed at established timepoints, and spleens were collected in RPMI 1640 medium. Spleen single-cell suspensions were made by passing splenic tissue through a 70-μm cell strainer in RPMI 1640 medium, and these were then treated with Red Blood Cell Lysis Buffer. Untouched B cells were isolated from spleen single-cell suspensions by using the mouse B Cell Isolation Kit (Miltenyi Biotec). B cell samples were then evaluated for purity (~97-98%, based on B220 expression assessed by flow cytometry) and stored in RNALater. Total RNA was extracted by using the RNeasy Mini kit (Qiagen). Total RNA (≥ 1ug) was sent to LC Sciences (Houston, TX) for Poly(A) RNA-Seq Sequencing Service. The total RNA quantity and purity were analyzed by the Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511). High-quality RNA samples with RIN number > 7.0 were used to construct sequencing library. mRNA was purified from total RNA using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat. e6150, USA, under 94℃ 5-7min). Then, the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were then used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat. m0209, USA), RNase H (NEB, cat. m0297, USA) and dUTP Solution (Thermo Fisher, cat. R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat. m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec, and then final extension at 72℃ for 5 min. The average insert sizes for the final cDNA librarys were 300±50 bp. At last, we performed the 2×150 bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (LC Sciences, Houston, TX, US) following the vendor's recommended protocol. 2×150bp paired-endsequencing (PE150)