title: Illumina HiSeq 4000 paired end sequencing; RNA-seq of splenic and LN B cells from WT and Gm40600 Tg mice with or without LPS stimulation
design: RNA-seq of splenic and LN B cells from WT and Gm40600 Tg mice with or without LPS stimulation
library_name: Sample 2_p
library_construction_protocol: Splenic lymphocytes were isolated from 9-week-old female wildtype (WT) mice and Gm40600 transgenic (Gm40600 Tg) mice (3 mice/group) using Ficoll (Ficoll-Paque Plus, GE Healthcre). B cells were sorted by B220 microbeads (Cat No. 130-049-501, Miltenyi Biotec, Germany) or stimulated for 3 days with 10 ug/ml LPS. Samples were washed twice with PBS and 1 mL of TRIzol per 1 x 107 cell pellets was added and frozen in -80℃. Mice were euthanized using CO2. Spleens and Lymph nodes (LNs) were cut using scissors.  B cells were sorted by B220 microbeads (Cat No. 130-049-501, Miltenyi Biotec, Germany) or stimulated for 3 days with 10 ug/ml LPS.  Wildtype (WT) mice and Gm40600 transgenic (Gm40600 Tg) mice were developed by Cyagen Co., Ltd (Guangzhou, China). Splenic and lymph nodes (LNs) lymphocytes were isolated from 9-week-old female wildtype (WT) mice and Gm40600 transgenic (Gm40600 Tg) mice (3 mice/group) using Ficoll (Ficoll-Paque Plus, GE Healthcre). B cells were sorted by B220 microbeads (Cat No. 130-049-501, Miltenyi Biotec, Germany) or stimulated for 3 days with 10 ug/ml LPS. Samples were washed twice with PBS and 1 mL of TRIzol per 1 x 107 cell pellets was added and frozen in -80℃. Data were aligned using STAR with options --outFilterScoreMinOverLread 0.4 --outFilterMatchNminOverLread 0.4, against the C3H/HeJ genome as a reference (ftp://ftp.ensembl.org/pub/release-89/fasta/mus_musculus_c3hhej/dna/Mus_musculus_c3hhej.C3H_HeJ_v1.dna_sm.toplevel.fa.gz).  Total RNA was extracted from 107 PBMCs using TRIzol reagent (Life Technologies).Total RNA from cells was purified using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands). All RNA content in the samples was measured with a NanoDrop®ND-1000 spectrophotometer, and the RNA quality was assessed using RNA 6000 NanoChips with the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina).
attributes.tag: Experimental Factor: genotype
attributes.value: Gm40600 Tg
attributes.tag: Experimental Factor: compound
attributes.value: none
attributes.tag: Experimental Factor: organism part
attributes.value: spleen