title: Proseq_HEK_WT_br3
organism: Homo sapiens
description: Protocols: To generate CRISPR/Cas9 PHF3 KO, gRNA targeting exon 3 was cloned between BbsI sites under the U6 promoter in the plasmid encoding Cas9-EGFP (PX45870). gRNA sequence for human PHF3 was 5'-TGATACTAGTACTTTTGGAC-3' and for mouse PHF3 5'-ATTCGGGTTCCTCGGCCGTC-3'. 48h after transfection with polyethylenimine (PEI; Polysciences), GFP-positive HEK293T cells were FACS-sorted and allowed to recover in culture for 4 to 7 days. Cells were subsequently FACS-sorted and GFP-negative cells were seeded 1 cell/well in 96-wells plates. After 14 to 20 days, surviving clones were expanded in culture, genomic DNA was isolated and PCR-amplified Cas9 target region was sequenced.To generate CRISPR/Cas9 PHF3ΔSPOC, one gRNA target site on either side of the SPOC domain was selected in such a way that the PAMs are within the deleted fragment (5'-TGGCTCGATTGAACTTCATC-3' and 5'-GGTCCATCAAAAGGCACAAG-3'). A 150 bp ssDNA repair template was designed which introduces an XhoI restriction site at the junction of the two breaks without shifting the reading frame or altering the amino acid sequence (5'-TTAGGCTTTTTAATGTCATTTTCTAGTCCAGAGATGCCTGGAACTGTTGAAGTTGAGTCTACCTTTCTGGCTCGAGTGCCTTTTGATGGACCTGGTAGGTATACGTTTTAATAATAGGATAAGAATGAAATAACATGGGAGGTGGGACCA-3'). S-phase synchronized HEK293T cells were electroporated with 10 µg of purified Cas9, 12 µg of each in vitro transcribed gRNA and 4 µM repair template. Genomic DNA from the clones was PCR-amplified to check for the deletion of 4.5 kb and digested with XhoI to ensure that the repair template was used for homologous recombination-mediated repair. The PRO-seq protocol was adapted from Kwak et al, 2013. To isolate nuclei, cells were resuspended in cold buffer A (10 mM Tris-Cl pH 8, 300 mM sucrose, 3 mM CaCl2, 2 mM MgAc2, 0.1% TritonX-100, 0.5 mM DTT), incubated on ice for 5 min and transferred to a dounce homogenizer. After douncing 25 times with the loose pestle, cells were centrifuged at 700 g for 5 min. The pellet was resuspended again in buffer A, centrifuged and resuspended in cold buffer D (10 mM Tris-Cl pH 8, 25% glycerol, 5 mM MgAc2, 0.1 mM EDTA, 5 mM DTT), flash frozen with liquid nitrogen and stored at -80°C. For each run-on, 107 HEK293 nuclei was mixed with 106 of Drosophila S2 nuclei (10% for spike-in normalization) in 100 µL buffer D and incubated at 30°C for 3 min with 0.025 mM biotin-11-NTPs and run-on master mix (5 mM Tris-Cl pH 8, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.2 units/µL SUPERase In, 0.5% Sarkosyl). Nascent RNA was isolated using TRIzol LS reagent according to the manufacturer's instructions, denatured at 65°C for 40 s, hydrolyzed using 0.2 M NaOH on ice for 20 min and neutralized with 1 volume of 1 M Tris-Cl pH 6.8. Buffer was exchanged with DEPC water using BioRad P-30 columns. Fragmented nascent RNA was subsequently enriched using Streptavidin M280 beads by rotating the samples for 20 min in binding buffer (10 mM Tris-Cl 7.4, 300 mM NaCl, 0.1% TritonX-100). Beads were subsequently washed twice with high-salt wash buffer (50 mM Tris-Cl pH 7.4, 2 M NaCl, 0.5% TritonX-100), twice with binding buffer and once with low-salt wash buffer (5 mM Tris-Cl pH 7.4, 0.1% TritonX-100). RNA was isolated from the beads using TRIzol reagent in two consecutive rounds and pooled together for ethanol precipitation. The RNA pellet was redissolved in DEPC H2O with 10 pmol of reverse 3' RNA adaptor and subjected to ligation using T4 RNA ligase I (NEB) at 16°C ON. RNA was isolated using Streptavidin M280 beads as previously described and 5' ends were repaired using Cap-Clip Acid Pyrophosphatase (Cellscript) and Polynucleotide Kinase (PNK, NEB) according to manufacturers' instructions. RNA was purified again with TRIzol and ethanol precipitation as before and subjected to 5' RNA adaptor ligation as for the 3' adaptor ligation. RNA was enriched by a third round of binding to Streptavidin M280 beads and TRIzol isolation. RNA was retro-transcribed using RP1 Illumina primer and SuperScript III (Invitrogen) to generate cDNA libraries. Libraries were then amplified using KAPA HiFi Real-Time PCR Library Amplification Kit (Peqlab) and Illumina primers containing standard TruSeq barcodes and a random octamere sequence. Amplified libraries were subjected to electrophoresis on 2.5% low melting agarose gel and amplicons from 200 to 300 bp were excised, purified from the gel using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-12-02T04:02:44Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-12-07T15:44:42Z
attributes.tag: External Id
attributes.value: SAMEA5159649
attributes.tag: INSDC center name
attributes.value: Max Delbrueck Center
attributes.tag: INSDC first public
attributes.value: 2019-12-02T04:02:44Z
attributes.tag: INSDC last update
attributes.value: 2018-12-07T15:44:42Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7501:Proseq_HEK_WT_br3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HEK293
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: kidney
attributes.tag: sample name
attributes.value: E-MTAB-7501:Proseq_HEK_WT_br3
attributes.tag: scientific_name
attributes.value: Homo sapiens