Computational protocol: A comprehensive analysis of AID's effects on the transcriptome and methylome of activated B cells

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Protocol publication

[…] Libraries were prepared as previously described. Two bases were trimmed from the 3′ end, and trimmed reads were aligned with Bowtie v0.12.7 against NCBI37 using the parameters “-l 15 -v 2 --best --strata -m 1”. Reads overlapping with sequences annotated in miRbase 18 were quantified with SeqMonk. [...] Genomic DNA was prepared from B cells using the Qiagen DNEasy kit. For each sample, 500 ng DNA was digested with 2 μl MspI for 18 h and purified by phenol/chloroform extraction. Following end repair, adenylation, and ligation of Illumina methylated adapters, products with size 200–350 bp were purified on agarose gels. Bisulfite conversion was performed twice with the Qiagen Epitect kit, and the library was amplified using Pfu Cx hotstart polymerase (Agilent).Sequencing on the Illumina HiSeq2000 yielded 47–54 million 50-nt reads per sample. Reads were trimmed using TrimGalore v0.2.2 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) 3 times sequentially with the parameters “-a AGATCGGAAGAGC”, then “-s 8 -a CGGTTCAG”, then “-s 8 AGCAGGAA”. Trimmed reads were aligned to the mouse genome (NCBI37) using Bismark v0.7.4 with Bowtie v0.12.7 with parameters “-l 20”. Analysis was performed using Seqmonk (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). Each position was used for analysis if it was covered at least 10x for all of the samples, and features were used if they contained at least 3 such positions. Features were defined using the Ensembl reference. Promoters were defined as −5 kb to +1 kb from the TSS. [...] Primers were designed using the Epidesigner tool (http://www.epidesigner.com/). Epityper assays were performed by the Weill Cornell Medical College Epigenomics Core. [...] All statistical analysis was performed using R software. Graphics were generated using the ggplot2 package. […]

Pipeline specifications

Software tools Trim Galore!, Bismark, Bowtie, SeqMonk, EpiDesigner, Ggplot2
Applications Miscellaneous, BS-seq analysis
Diseases Deficiency Diseases
Chemicals Cytidine