GSM7656488: RNA-seq_GATA3_KO_Naive_rep4; Mus musculus; RNA-Seq
Detailed procedures for ChIP-Seq: 1. Three plates (15-cm) of HeLa or SW-13 cells cultured in 20 ml of DMEM containing 10% FCS and antibiotics were stimulated with 500 units/ml of IFN-α for 2 hrs. 2. Add HCHO to 1% of each plate and incubate at 37°C for 10 min; Stop the crosslinking by aspirating the medium. Wash the cells twice with 10 ml of 1xPBS. 3. Aspirate the buffer (keep 0.5 ml in the plate). Harvest cells by scraping. Wash the plate once with 0.5% BSA/PBS. Combine the cells from the three plates. 4. Spin down the cells at 3000rpm, 3 min, 4oC. 5. Remove supernatant and resuspend the cells in 2.5 ml of 1 x TE + 1 mM PMSF + 1 x proteinase inhibitor cocktail. For anti-AcH3 or anti-AcH4 CHIP, add sodium butyrate to 5 mM. Sonicate 20 x 20 seconds in ice-water bath with power setting at 5 with an Ultrasonic Processor XL (HEAT System) to break chromatin to about 0.3-05 kb. 6. Adjust to RIPA buffer + 200 mM NaCl by adding 0.28 ml of 10% TX100, 28 μl of 10% Na-Deoxycholate, 28 μl of 10% SDS, 110 μl of 5M NaCl. Mix well. Incubate for 5 min on ice. Spin in a microfuge, 13K rpm, 10 min, 4oC. 7. Collect supernatant. 8. Add 4 μg of the antibodies to 40 μl of Dynabeads protein A beads. Incubate at 4°C 4- 6hrs with rotation. Wash away antibody by 1xPBS. 9. Add 500 μl of the chromatin ( sup.) to the beads+antibody. Incubate at at 4°C overnight with rotation. 10. Wash the beads twice with RIPA buffer; twice with RIPA buffer (+0.3M NaCl); twice with LiCl buffer (1xTE, 0.25M LiCl, 0.5% NP40, 0.5% Na-Deoxycholate); and twice with 1xTE. 11. Resuspend the beads in 100 μl of 1xTE, 2.5 μl of 10% SDS, 5 μl of 10 mg/ml proteinase K, mix well, incubate at 65oC, overnight. Also treat 100 μl of the input chromatin in the same way. 12. Collect sup. Wash the beads once with 100 μl of 1xTE. Combine the supernatants. Extract 1 x with Phenol-CHCl3. 13. Precipitate DNA with 2 μl of 20mg/ml glycogen, 20 μl of 3M NaOAc pH5.2, 500 μl of ethanol. Incubate 10 min on dry ice. Spin 20 min at 4 °C in a microfuge. Remove sup, wash pellet 1 x with 70% ethanol, air dry, resuspend in 30 μl of 1 x TE. Use 3 μl for one PCR reaction. 14. The concentration of the input DNA is determined by spectrophotometer. RIPA buffer: 1X In 1X TE 0.1% SDS 0.1% Na deoxycholate 1% Triton X100.                                                                                                                                                                                                                                   For RNA-Seq, five thousand cells were sorted into 700μl of QIAzol Lysis Reagent (miRNAeasy Micro Kit (QIAGEN, Cat#217084). Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat#79254) was performed, followed by elution with 10μl of RNase-free water. Total RNA from 1K cells was reverse transcribed by SuperScript II (Invitrogen, Cat#18064-014) with oligo-dT and LNA-containing TSO primers in a final reaction volume of 10μl using the condition: 42°C for 90min, 10 cycles of 50°C 2min to 42°C 2min, 70°C for 15min and hold at 4°C. cDNA was pre-amplified by PCR using KAPA HiFi HotStart ReadyMix (KAPABIOSYSTEMS Cat#KK2602) with IS PCR for 12 cycles in 25μl. PCR products were purified by Ampure XP beads (Beckman Coulter, Cat#A63881) and eluted with 17.5μl Nuclease-free water as described for Smart-seq2 method (Picelli et al., 2014). PCR pre-amplified cDNA (40μl) was sonicated to 200~400bps by Bioruptor Pico (Diagenode) for 15 cycles (30″ on and 30″ off). Sonicted cDNA was blunt-ended by End-It DNA-Repair kit (Epicentre, Cat#ER81050), ligated to “Y” adaptor and PCR-amplified for 12 cycles and sequenced as described for the scDNase-seq libraries. For Hi-TrAC, cells were fixed with 1% Formaldehyde at room temperature for 10 minutes, and then incubated with the transposase complex to bridge interacting chromatin at 37 °C for 2 hours. Genomic DNA was purified after reverse cross-linking. 1. Repair DNA ends using the Epicentre DNA END-Repair kit (Cat. No. ER0720, Epicentre Biotechnologies, Tel 800-284-8474) This step generates blunt-ended DNA. 1-34 ul DNA (0.1 to 5 ug) 5 ul 10x End repair buffer (330 mM Tris-acetate, pH7.8, 660 mM potassium acetate, 100 mM magnesium acetate, 5 mM DTT) 5 ul 2.5 mM each dNTPs 5 ul 10 mM ATP x ul H2O to adjust the reaction volume to 49 ul 1 ul End-Repair Enzyme mix (T4 DNA Pol + T4 PNK) Incubate 45 min at room temperature. Purify the DNA using MinElute Reaction Cleanup Kit and protocol, eluting in 32 ul of EB. 2. Add “A” to 3' ends 32 ul DNA from above 5 ul 10x Klenow buffer 10 ul 1 mM dATP 3 ul 5 U/ ul Klenow Exo- Incubate for 30 min at 37°C. Purify the DNA as before. Eluting in 22 ul of 1x EB. 3. Linker ligation (two mixed linkers). The oligo mix should contain 10x molar excess of the DNA templates. 23 ul DNA (300ng) 3 ul 10x T4 DNA ligase buffer 1 ul Index PE Adapter Oligo Mix (1:10 diluted) 3 ul T4 DNA ligase (400 units/ul) 2 Incubate for 30 min at room temperature. Purify the DNA as before. Eluting in 20 ul of 1x EB. 4. Size selection-2% E-Gel Load 20 ul linker ligated DNA onto a 2% E-Gel (Invitrogen). Cut the gel around 200-400 bp region (DNA will not be visible). Extract the DNA using MinElute gel extraction kit (every 900ul of QG Buffer add 400ul of 2-Propanol). Elute in 22 ul EB. (Note: (1) Dissolve gel slices at room temperature with frequent mixing, but not at elevated temperature. This helps to reserve AT-rich DNA that can be easily denatured at higher temperature and gets lost at column binding step. (2) NEVER run PCR-amplified samples of Step 7 with the linker-ligated products of Step 5 together on the same gel because the latter can be contaminated!). 5. Amplify the DNA using Solexa primers and enzyme mix (using half amount of the reagents and run a 25 ul reaction). 18 ul of DNA 20 ul of master mix 1 ul of PCR primer InPE1.0 (2x diluted) 1 ul of PCR Primer Index<#> (2x diluted) Total volume: 50 ul Denature at 98°C for 30 sec. 98°C, 10”; 65°C, 30”; 72°C, 30”. Try 18 cycles first, check 2.5 ul of product on 1.8% gel. If the band is not clearly visible, do 3 more cycles. Check again. 6. Purify the amplified products on 2.5% agarose gel. Excise the band near 220 bp and purify the DNA using QIAquick gel extraction kit. Measure the DNA concentration using Qubit fluorometer. (Note: Dissolve gel slices at room temperature with frequent mixing, but not at elevated temperature. This helps to reserve AT-rich DNA that can be easily denatured at higher temperature and gets lost at column binding step). For Hi-TrAC libraries, gaps in purified genomic DNA was repaired with T4 DNA polymerase. Genomic DNA was digested with MluCI and NlaIII restriction enzymes, and biotin labeled DNA fragments was enriched by streptavidin beads. Perform adapter ligation with T7 ligase on beads, and then run PCR to amplify and add index to libraries.