GSM4851744: RNA-seq_Pax5_KO+pMiG_rep2; Mus musculus; RNA-Seq
For RNA-seq: Total RNA was isolated by use of RNAeasy Micro Kit (Qiagen, Hilden, Germany) according to  manufacturer's recommendations. For ChIP-seq: Cells for transcription factor ChIP were fixed at room temperature with DSG for 30 min followed by 1% formaldehyde/PBS for 10 minutes. Reaction was then quenched by adding glycine to 0.125 M. Cells were washed  pelleted and snap-frozen and stored at -80°C until ready for ChIP or used immediately for ChIP.   Nuclei for ChIP were isolated  by 10 min incubation in Nuclei Isolation buffer (50 mM Tris-pH 8.0, 60 mM KCl, 0.5% NP40) + protease inhibitor cocktail (PIC) (Roche) on ice. Pelleted nuclei were dissolved in Lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8))+ PIC and sonicated on a Bioruptor (Diagenode) max power for 30s followed by 30 s rest. Sonication was followed by pelleting of debris and the supernatant was transferred to new tube and chromatin was diluted 5X in Dilution Buffer (1% Triton, 2mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8) + PIC). Anti-bodies were hybridized to ProteinA or G Dynabeads®  (LifeTechnologies)  and added to the diluted chromatin. ChIP was performed over night at 4°C. and subsequently washed (1 time with 500 μl Low Salt Immune Complex Wash Buffer, 1 time with 200 μl High Salt Immune Complex Wash Buffer, 1 time with 200 μl LiCl Immune Complex Wash Buffer, 2 times with 200 μl TE buffer) and eluted for 4 h at 65°C ( 20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, 100 μg RNase A and 50 μg proteinase K) treated and finally cleaned up using Zymo ChIP DNA Clean & Concentrator before ChIP-seq libary preparation.PLAC-seq was carried out similar to previously reported (Ahsberg et al., 2019) with minor modifications. 15M FL derived Wt or Ebf1-/- pro-B cells that had been cross-linked with 1% formaldehyde for 5 min were thawed on ice for 5 minutes, re-suspended in ice cold lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP-40 and  Roche protease inhibitors – 11697498001 (PIC)) and incubated on ice for 15 min. Samples were spun for 5 min @ 4°C at 2,500 x g and cell pellet was washed in ice-cold lysis buffer + PIC. The pellet was resuspended in 0.5% SDS and incubated @ 62°C for 10 min. The SDS reaction was quenched by addition of water and 10% Triton X-100 followed by 15 min incubation @ 37°C. The samples were digested with 40U MboI restriction enzyme with addition of 25 µl NEBuffer2 followed by 2h incubation @ 37°C with shaking (900 RPM). After restriction enzyme digestion the samples were incubated for 20 min @ 62°C to inactivate MboI followed by a cool-down to RT. A fill in reaction was conducted using 0.3 mM Biotin-14-dATP (ThermoFisher, 19524016), 0.3 mM dCTP, 0.3 mM dTTP, 0.3 mM dGTP and 40U Klenow (NEB, M0210) @ 37°C for 1.5h with shaking (900 RPM). Next a Ligation master mix (1x T4 ligation buffer (NEB, B0202), 1% Triton-X 100, 120 ug BSA (NEB, B9000), 4000 U T4 DNA ligase (NEB, M0202)) was added and samples were incubated @ RT with rotation for 2h. The samples were centrifugated 2,500xg 5min @ 4°C, supernatant was removed and cell pellet was resuspended in 250 µl ChIP SDS-lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8)) + PIC followed by sonication on a Covaris ME220 for 6 min (Peak power = 75, cycles per burst = 1000, Duty Factor = 15%). Sheared chromatin was centrifuged for 10 min @ 4°C, 13000 rpm and supernatants were transferred to new tubes and diluted in 750 µl 1xHBSS + 1 ml 2x RIPA (20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 2% Triton X-100, 0.1% SDS, 0.2% sodium deoxycholate, 200 mM NaCl) + PIC. 1% input was removed from supernatant, and 10 µg H3K4-Me3 (Millipore, 07-473) antibody pre-adsorbed to 60 µl Protein-G dynabeads in PBS/0.5% BSA were added to the remaining supernatant and incubated @ 4°C overnight with rotation. Samples were washed as follows: 2 times with 1 ml Low Salt Immune Complex Wash Buffer (0.1% SDS, 1 % Triton X-100, 2 mM EDTA, 50 mM Tris-HCl pH8, 150 mM NaCl), 2 times with 1 ml High Salt Immune Complex Wash Buffer (0.1% SDS, 1 % Triton X-100, 2 mM EDTA, 50 mM Tris-HCl pH 8.0, 500 mM NaCl), 1 time with 1 ml LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% Igepal-CA630, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), 2 times with 1 ml TE buffer (10 mM Tris–HCl, pH 8.0, 10 mM EDTA) followed by elution of chromatin from magnetic beads with two rounds of 100 µl elution buffer (1%SDS, 100 mM NaHCO3) with shaking (1500 RPM) @ RT. Supernatants were transferred to new tubes and chromatin complexes were reverse cross-linked overnight @ 65°C with the addition of 250 mM NaCl, 100 μg RNase A (ThermoFisher, EN0531) and 50 μg proteinase K (ThermoFisher, AM2546) followed by Zymo Research ChIP DNA Clean & Concentrator (BIOSITE-D5205) clean up. 25 μL of Streptavidin T1 beads (Thermo Fisher, 65601) were washed with Tween Wash Buffer (5 mM Tris-HCl pH 8, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20) then resuspended in 50 μL of 2x Biotin Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2M NaCl). Beads were added to the samples and incubated at room temperature for 15 minutes with shaking. RNA-seq: Libraries were constructed using NuGEN's Ovation Ultralow Library systems (NuGEN Technologies, San Calros, CA) and were subsequently subjected to 76 cycles of NextSeq500 sequencing (Illumina, San Diego,CA). ChIP-Seq: Libraries were constructed using Illumina Truseq Nano DNA library preparation kit or by addition of Bioo-scientiffic Nextflex barcodes following standard procedures. Libraries were run on the Illumina NexSeq500.   For PLAC-seq libraries: After capture on magnetic beads, they were placed on a magnet and supernatant was discarded. Samples were washed twice by adding 500 μL of Tween Wash Buffer and incubated at 55°C for 2 minutes shaking followed by a 1 time wash in 100 µl 1x T4 DNA ligation buffer. Beads were collected on a magnet and resuspended on 100 µl end-repair mastermix (0.5 mM dNTPs (VWR, E636-40UMOLE), 12U T4 DNA Polymerase (NEB, M0203), 50U T4 Polynucleotide Kinase (NEB, M0201), 5U Klenow (NEB, M0210), 1x NEB T4 DNA ligase buffer) followed by incubation @ RT for 30 min with shaking (900 RPM). 300 µl Tween Wash Buffer was added and beads were placed on a magnet and supernatant was discarded. Samples were washed twice by adding 500 μL of Tween Wash Buffer and incubated at 55°C for 2 minutes shaking followed by a 1 time wash in 100 µl 1x NEB2 buffer. magnetic beads were collected on a magnet and resuspended on 100 µl A-tailing mastermix (0.5 mM dATP (ThermoFisher), 25U Klenow Exo- (NEB, M0212), 1x NEB2 buffer) and incubated @ 37°C for 30 min with shaking (900 RPM). 300 µl Tween Wash Buffer was added and beads were placed on a magnet and supernatant was discarded. Samples were washed twice by adding 500 μL of Tween Wash Buffer and incubated at 55°C for 2 minutes shaking followed by a 1 time wash in 100 µl 1x Fast-Link ligation buffer (Epicenter, LK0750H ) followed by NEXTflex DNA barcode ligation  (BIOO scientific) using the Fast-link ligation kit (Epicenter, LK0750H). 300 µl Tween Wash Buffer was added and beads were placed on a magnet and supernatant was discarded. Samples were washed twice by adding 500 μL of Tween Wash Buffer and incubated at 55°C for 2 minutes shaking followed by a 1 time wash in 100 µl 10mM Tris-HCl pH 8.0, resuspended in 45 µl and a 1:1000 dilution was made for qPCR determination of number cycles needed for final PCR amplification. After final PCR, T1 streptavidin beads were collected on a magnet, supernatant transferred to new tubes and Ampure XP beads (x0.8 sample volume) were used to clean up libraries. PLAC-seq libraries were subject to 2x 75 cycles of paired-end sequencing on a NextSeq500.