title: Illumina HiSeq 4000 paired end sequencing; RNA-seq of splenic B cells derived from WT and Itch cKO mice
design: RNA-seq of splenic B cells derived from WT and Itch cKO mice
library_name: Sample 1_p
library_construction_protocol: Splenic lymphocytes were isolated from 7-9-week-old female itchf/f wildtype (WT) mice and B-cell specific Itch knocked-out (CD19creItchf/f) 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). 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 were cut using scissors.  B cells were sorted by B220 microbeads (Cat No. 130-049-501, Miltenyi Biotec, Germany). B cells were not cultured in vitro. 1 mL of TRIzol per 1 x 107 cell pellets was added and frozen in -80℃.  CD19cre mice and Itchf/f mice were developed by Cyagen Co., Ltd (Guangzhou, China). CD19cre mice and Itchf/f mice were mated to get Itchf/f (WT) and CD19creItchf/f (Itch cKO). Splenic lymphocytes were isolated from 7-9-week-old female WT mice and Itch cKO 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). 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 B cells 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: wild type genotype