Computational protocol: IL 2 imprints human naive B cell fate towards plasma cell through ERK/ELK1 mediated BACH2 repression

Similar protocols

Protocol publication

[…] RNA was extracted using RNeasy microkit (cat no. 74004 from Qiagen) and reversed transcribed into cDNA with Superscript II (Invitrogen). QRT-PCR was performed using the TaqMan Gene Expression Master Mix and run on the Step One Plus Real-time PCR System from Applied Biosystems. All TaqMan primers (Applied Biosystems) used in this study are listed in Supplementary Table  Gene expression levels were quantified using HPRT1 as endogenous control. The 2 exp(−ΔΔCt) method was used to determine the relative expression of each gene.For single-cell QRT-PCR experiments, gene expression levels for the 16 selected Taqman assays within single-cell cDNA were measured with qPCR on 96.96 Dynamic Array IFC using the Fluidigm BioMark HD system. A total of 285 single cells from C1 captures were profiled using Dynamics Array IFC. Hierarchical clustering was performed on R version 3.2.4 with ComplexHeatmap (R package version 1.6.0), fluidigmSC (Fluidigm SINGuLAR Analysis Toolset, R package version 3.5.2), dendextend, amap (R package version 0.8–14) packages. We used a conservative Ct of 24 as LOD based on published guidelines . Gene expression was defined on a log2 scale as: log2 expression = LOD-Ct. Samples were clustered by the Euclidean distance, and genes were clustered by the pearson distance. Heatmap was generated with the ‘global_z_score’ (normalisation by the expression value with the global mean and the global standard deviation). [...] MatInspector, TFBIND, TFSEARCH (http://cbrc3.cbrc.jp/papia/howtouse/howtouse_tfsearch.html), PROMO and comparative genomics tool rVISTA were used for the identification of regulatory elements in human BACH2 locus. BLAST program (NCBI) was used to search for alignment. [...] All oligonucleotides used in the construction of the BACH2 luciferase reporter plasmids were designed using Primer3 and synthesised by Eurogentec. The list of cloning and sequencing primers is available in Supplementary Table . DNA insert sequences were amplified by PCR using Q5 High-Fidelity DNA polymerase (NEB) with primers containing restriction sites followed by PCR product purification (NucleoSpin Gel and PCR Clean-up, Machery-Nagel). Purified insert DNAs together with the appropriate expression vectors were then restriction digested using corresponding enzymes (CutSmart restriction endonucleases, NEB), purified and ligated together using T4 DNA ligase (Roche). The reporter vectors implemented for DNA insertion were the basic vector pNL1.1[Nluc] and minimal promoter vector pNL3.1[Nluc/minP] that both encoded the NanoLuc luciferase reporter gene (Promega). The pGL4.50[luc2/CMV/Hygro] vector (Promega) encoding the Firefly luciferase reporter gene luc2 (Photinus pyralis) was used for transfection efficiency. Escherichia coli cells (MAX Efficiency DH5α Competent cells, Invitrogen) were transformed with the recombinant plasmid DNA and individual colonies were then screened for the presence of the DNA insert by PCR (Taq DNA Polymerase with ThermoPol Buffer, NEB). Positively identified clones were sent for Sanger sequencing analysis. NCBI BLAST confirmed the absence of mutations. Finally, the selected plasmid DNA clones were further expanded and purified (NucleoBond Xtra Midi Plus EF, Machery-Nagel).The luciferase reporter containing the BACH2 minimal promoter (−725; +146), pNL1.1/minPBACH2, was constructed through PCR amplification from tonsil B cell DNA as previously described by ref. , followed by NheI/XhoI restriction digestion and ligation into the pNL1.1 [Nluc] vector.The luciferase reporter vector containing the 228 bp (+1265; +1493) BACH2 enhancer (Enh), pNL1.1/minPBACH2/Enh, was constructed through PCR amplification from the PAC clone RP1-104D1 followed by XhoI restriction digestion and downstream ligation into the BACH2 minimal promoter construct pNL1.1/minPBACH2. The enhancer was then sub-cloned from pNL1.1/minPBACH2/Enh and ligated into the XhoI site of the independent minimal promoter vector pNL3.1[Nluc/minP] to generate minPPNL3.1/Enh. All enhancer inserts were screened by PCR to identify sequence ligations in both the 5′-3′ and 3′-5′ orientation. Sequencing confirmed the orientation of inserts.The remaining luciferase reporter constructs containing fragmented sequences of the BACH2 enhancer, namely Enh80 (+1265/+1366), Enh122 (+1388/+1493) and Enh115 (+1265/+1381) were generated by PCR amplification with primers containing XhoI restriction sites at the 5′end and ligation into pNL1.1/minPBACH2. pNL1.1/minPBACH2/Δ21nt-Enh was generated by sub-cloning the Enh122 sequence by blunt ended ligation into the EcoRV site of the pNL1.1/minPBACH2/Enh80 vector. All fragmented enhancer inserts were screened by PCR to identify sequence ligations in both the native 5′-3′ and reverse 3′-5′ orientation.For preparation of the mutant Mut-PU1bs and Mut-ELK1bs forms of the enhancer, pNL3.1/Enh was implemented as DNA template for PCR site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, cat no. E0554 from NEB). The presence of the substitution mutations within the enhancer was confirmed by sequencing. The mutated enhancer sequences were then sub-cloned by XhoI restriction digestion into the XhoI site of pNL1.1/minPBACH2 to generate pNL1.1/minPBACH2/Mut-PU1bs and pNL1.1/minPBACH2/Mut-ELK1bs. [...] Three experiments from independent blood donors were analysed. RNA extractions were performed with the NucleoSpin RNA XS kit (cat no. 740902.250 from Macherey-Nagel). QC, library preparation and sequencing were performed by Helixio company (Clermont Ferrand, France). Briefly, libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (cat no. RS-122-2101 from Illumina) and samples were sequenced on an Illumina NextSeq 500 using 75-bp single-end reads (NextSeq 500 High Output v2, Illumina). Quality of sequencing data was monitored by FastQC. Residual adapters from sequencing were trimmed using cutadapt 1.0. Potential PCR duplicates were removed using SAMtools 1.3. Reads were then aligned on the GRCh38 human genome using STAR 2.4.2a. Differential expression on filtered genes (HTSfilter 1.7.1), were performed using DESeq2 in R 3.3.1. Genes were declared differentially expressed with a false discovery rate < 5%. For heatmap, genes differentially expressed with fold change >1.4 in CD25hi and siBACH2 condition compare to CFSElo population was ploted. Log2 normalised read counts from DESeq2 were used to generate the representation with gplots 3.0.1, and RColorBrewer 1.1–2. Hierarchical clustering was generated using Euclidean distance matrix with complete linkage. […]

Pipeline specifications

Software tools Primer3, BLASTN, FastQC, cutadapt, SAMtools, HTSFilter, DESeq2, gplots
Applications RNA-seq analysis, qPCR
Organisms Homo sapiens