E-MTAB-8273:Sample 2_p
Illumina HiSeq 4000 paired end sequencing; RNA-seq of splenic B cells derived from WT and Itch KO mice
RNA-seq of splenic B cells derived from WT and Itch KO mice
Sample 2_p
Splenic lymphocytes were isolated from 7-9-week-old female wildtype (WT) mice and Itch knocked-out (KO) 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℃.  Wildtype (WT) mice and Itch knocked-out (KO) mice were developed by Cyagen Co., Ltd (Guangzhou, China). Splenic lymphocytes were isolated from 7-9-week-old female wildtype (WT) mice and Itch knocked-out (KO) 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 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).
Beijing Institute of Basic Medical Sciences
E-MTAB-8273:Sample 2_p
Experimental Factor: genotype
Itch KO