title: Illumina HiSeq 4000 paired end sequencing; RNA-seq of CSMD1 enhancer CRISPRi in LUHMES cells and differentiated neurons
design: RNA-seq of CSMD1 enhancer CRISPRi in LUHMES cells and differentiated neurons
library_construction_protocol: RNA was extracted using the RNeasy Mini Kit (QIAGEN) and cDNA was produced by employing the Transcriptor First Strand cDNA Synthesis Kit (Roche) following the manufacturer´s procedures. RT-qPCR reactions were performed with SYBR Green Master Mix (Thermo Fisher) and run on an Applied Biosystems QuantStudio 12K Flex Real-Time PCR machine. Relative gene expression values were calculated with the –ΔCt method, using TATA-Box Binding Protein (TBP) as a house-keeping gene for normalization. LUHMES cells were purchased from ATCC (CRL-2927). Cells were cultured as previously described (Scholz et al, JNC 2011). Briefly, cells were attached on pre-coated multi-well plates (50 ug/mL poly-L-ornithine and 1 ug/mL fibronectin - Sigma) and grown in proliferation medium: Advanced DMEM/F-12 plus N-2 Supplement (Thermo Fisher), 2 mM L-glutamine (Sigma) and 40 ng/mL recombinant basic fibroblast growth factor (R&D Systems). Cells were differentiated into neurons for 7 days using standard differentiation medium: Advanced DMEM/F-12 plus N-2 Supplement, 2 mM L-glutamine, 1 mM dibutyryl cAMP (Sigma), 1 ug/mL tetracycline (Sigma) and 2 ng/mL recombinant human GDNF (R&D Systems).  293FT cells were transfected with 3rd generation system lentiviral plasmids (pMDLg/pRRE, pRSV-Rev and pMD2.G; Addgene #12251, 12253 and 12259, respectively) to generate viral particles using the PEIpro reagent (Polyplus-Transfection). An additional plasmid consisting of KRAB-dCAS9 (Addgene 118155) was co-transfected with the previous ones. These vectors were used to perform CRISPRi Culture medium was changed the following day of transfection (Day 1) and virus was collected 48 hours after this (Day 3). Lentiviral particles were concentrated ~100 times using Lenti-X Concentrator (Takara). LUHMES cells were seeded in a 12-well-plate format and immediately transduced with 30 ul of virus concentrate. Culture medium was changed the following day and drug selection started 3 days after transduction and continued until control un-transduced cells were completely dead. 4.5 ug/mL of blasticidin (KRAB-dCAS9) treatment was used for LUHMES selection. RNA was extracted using the RNeasy Mini Kit (QIAGEN) and cDNA was produced by employing the Transcriptor First Strand cDNA Synthesis Kit (Roche) following the manufacturer´s procedures. RT-qPCR reactions were performed with SYBR Green Master Mix (Thermo Fisher) and run on an Applied Biosystems QuantStudio 12K Flex Real-Time PCR machine. Relative gene expression values were calculated with the –ΔCt method, using TATA-Box Binding Protein (TBP) as a house-keeping gene for normalization. Each RNA sample were quantified using Qubit and run on a Tapestation to assess sample integrity. mRNA was isolated from total RNA using oligo dT beads. After fragmentation the samples were converted to double stranded DNA and ligated to Illumina adapters. The second strand cDNA was then degraded by the UNG enzyme providing directional information to the library. Indexing of each sample was performed by PCR. Each library was quantified by Qubit and run on a Tapestation to assess fragment distribution and presence of artefacts. The libraries were then pooled and the resulting pool was quantified by qPCR prior to sequencing.