GSM2461728: RNA-seq WT B-1a [17253]; Mus musculus; RNA-Seq
Rneasy Plus Mini Kit, Dynabeads mRNA purification kit For strand specific RNA-sequencing, RNA was isolated with an RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by fragmentation by heating at 94 °C for 3 min (in fragmentation buffer). The fragmented mRNA was used as template for first-strand cDNA synthesis with random hexamers and a Superscript VILO cDNA Synthesis kit (Invitrogen). The second-strand cDNA was synthesized with 100 mM dATP, dCTP, dGTP and dUTP in the presence of RNase H, E. coli DNA polymerase I and DNA ligase (Invitrogen). Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module. For strandspecific RNA-sequencing, the uridines present in one cDNA strand were digested with uracil-Nglycosylase (New England Biolabs) as described10 followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems). Completed libraries were quantified with the Agilent Bioanalyzer dsDNA 1000 assay kit and Agilent QPCR NGS Library Quantification kit. For analysis of Bhlhe41 binding, Chromatin from 6x107 B-1a cells was prepared using a lysis buffer containing 0.25% SDS. The cells were subjected to ChIP as described11 with minor modifications. Anti-V5 agarose beads (Sigma- Aldrich) were used for precipitation. Sequencing libraries were prepared with the NEBNext Quick Ligation Module, NEBNext Endrepair Module and NEBNext dA-tailing module or NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems) and Illumina deep sequencing.  Open chromatin regions were mapped in sorted Vh12/Vk4 transgenic WT and DKO B-1a cells from the spleen of bone marrow chimeras by ATAC-seq, as described by Buenrostro et. al. with the following modification. The nuclei were prepared by incubation of cells with nuclear preparation buffer (0.30 M sucrose, 10 mM Tris, pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.1% NP40, 0.15 mM spermine, 0.5 mM spermidine and 2 mM 6AA) before the Tn5 treatment (3 × 104 cells with 4 ?l of the Nextera Tn5 transposase).
GEO Accession
GSM2461728