title: GSM3137743: Definitive endoderm small fraction Rep 2; Mus musculus; OTHER
library_construction_protocol: We provide a detailed protocol describing how to perform isolation of srHC and how to obtain samples ready to be used for libraries preparation starting from low cell number samples.    A) Related publications: (38–40)    B) Reagents:  • Agencourt® AMPure® XP (Beckman Coulter, cat. no. A63881)   • 80% EtOH  • Buffer EB (10 mM Tris-Cl, pH 8.5)  • Glycine (2M solution) (Fisher BioReagents BP381-1)  • nuclear lysis buffer (50mM Tris-Cl pH 8.0, 10mM EDTA, 1% SDS - Lonza, #51213, supplemented with protease inhibitors)  • TE buffer (10 mM Tris·Cl, pH 8.0, 1 mM EDTA)  • RNase A (Roche, #10109169001)  • Glycogen (Roche, #10901393001)    C) Equipment:  • COVARIS S220 (Focused Ultrasonicator S220)  • microTUBE AFA Fiber Pre-Slit Snap-Cap (COVARIS, cat. #520045)  • DynaMag™-2 Magnet (Thermo Fisher Scientific, #12321D)  • Agilent 2100 Bioanalyzer system (Agilent Technologies, G2939AA)  • Eppendorf Tubes    D) Procedure:  • Cell Preparation  1. Prepare thirty thousand (30,000) cell aliquots in 500 ul PBS from sorted cells.  2. Add 13.5 ul of 37% formaldehyde (Fisher Chemical F79-500) per 500 ul sample.   3. Incubate samples 10 min at RT on rotating platform.  4. Stop crosslinking reaction by adding 36 ul of 2M glycine (Fisher BioReagents BP381-1) to each sample.  5. Incubate samples for 5 min incubation at RT on rotating platform.  6. Centrifuge samples at 2000 RPM for 15 min at 4 °C.  7. Wash cells twice in cold PBS (use 10 ul of FBS - Hyclone, SH30071, in 500 ul of PBS). For each wash, centrifuge samples at 2000 RPM for 5 min at 4 °C. Slowly aspirate supernatant with pipetman, leaving approximately 10 ul of solution behind.  8. Resuspend the cells in 130 ul nuclear lysis buffer (50mM Tris-Cl pH 8.0, 10mM EDTA, 1% SDS - Lonza, #51213, supplemented with protease inhibitors - 1 tablet Complete Mini Protease inhibitor cocktail tablets - Roche, #04693159001-  per 10 ml Buffer).    • Chromatin Sonication  1. Sonicate chromatin using COVARIS S220 following these parameters:   Peak Power  200   Duty Cycle  5%   Burst/Cycle  200   Time   10'    • DNA purification  1. Adjust sample volume to 200ul with nuclear lysis buffer.  2. Reverse crosslinking by incubating samples at 65°C/18hours on heat block`.  3. Add 200ul TE buffer (10 mM Tris·Cl, pH 8.0, 1 mM EDTA), 8uL of 10mg/ml RNase A (Roche, #10109169001) to the samples.   4. Incubate the samples for 2hr at 37 °C.  5. Add 4ul, 20mg/ml Proteinase K (Roche, #03115828001) to the samples.   6. Incubate the samples for 2hr at 55 °C.  7. Purify DNA using conventional phenol:chloroform:isoamyl extraction. Briefly, add 400 ul phenol:chloroform:isoamyl mix to each sample. Mix and quickly vortex the samples.  8. Spin down the samples at max speed for 5 min at RT.  9. Transfer 350 ul of aqueous phase to a new eppendorf  10. Add 16 ul 5M NaCl, 1.5ul 20mg/ml glycogen (Roche, #10901393001) and 800 ul 100% EtOH to each sample.  11. Mix the samples thoroughly by inverting the tube 10-15 times.  12. Incubate the samples are incubated overnight at -80 °C.  13. Centrifuge the samples for 10 min at max speed at 4 °C.  14. Wash the samples with 500 ul 80% EtOH.  15. Centrifuge the samples for 10 min at max speed at 4 °C.  16. Air-dry sample pellet at RT 5 min and resuspend it in 21 ul EB buffer.  17. Save 1 ul purified DNA for checking sonication at the Bioanalyzer (Agilent Technologies, G2939AA) in parallel with further processed samples.    • Sonication-resistant chromatin selection  1. Add 0.7X AMPure XP beads (Beckman Coulter, A63881) to the each sample (14ul in 20ul initial solution). Please note that the amount of AMPure XP beads indicated has been optimized for 3*103 cell samples. Samples of different cell number might require adjustment in AMPure XP beads:volume ratio.  2. Mix samples thoroughly by pipetting 10-12 times.  3. Incubate samples at RT for 5 min.  4. Transfer eppendorf tubes to a magnetic rack (Thermo Fisher Scientific, #12321D) and beads are allowed to separate for 5 min at RT.  5. Transfer clear solution to a new eppendorf tube (this fraction is the small, sonication-sensitive fragments).  6. Wash beads (to which large, sonication-resistant fragments are bound) with 200ul EtOH 80% while tubes are maintained on rack.   7. Incubate samples for 30 sec with EtOH.  8. Remove EtOH and wash once more each sample.  9. Air-dry beads 5 min while tubes are kept on the rack.   10. Remover tubes from the rack  11. Elute the beads in 20 ul TE buffer, by pipetting thoroughly.  12. Save 1 ul purified DNA before further sonication to check samples size at the Bioanalyzer (Agilent Technologies, G2939AA) in parallel with further processed samples.  13. Incubate the samples are incubated 5 min at RT.   14. Place the samples on the magnetic rack (Thermo Fisher Scientific, #12321D).  15. Incubate the samples for 5 min.  16. Transfer clear solution to a new tube (this fraction is the large, sonication-resistant portion of DNA).    • Sonication of large DNA fragments  1. Adjust sample volume to 130ul adding TE buffer.   2. Sonicate the samples using COVARIS S220 (Focused Ultrasonicator S220) following these parameters:  Peak Power  175  Duty Cycle  10%  Burst/Cycle  200  Time   6'    3. Adjust sample volume to 200ul with TE buffer. DNA precipitation can be applied also to small, sonication-sensitive sample.  4. Add 1.0 ul 20mg/ml glycogen (Roche, #10901393001), 1/10 volume of sodium acetate (3 M, pH 5.2) and 3.0X volume EtOH 100% to each sample  5. Mix the samples thoroughly by inverting the tube 10-15 times.  6. Incubate the samples 4h at -80 °C.  7. Centrifuge the samples for 10 min at max speed at 4 °C.  8. Wash the samples with 500 ul 80% EtOH.  9. Centrifuge the samples for 10 min at max speed at 4 °C.  10. Air-dry sample pellet at RT 5 min and resuspend it in 11 ul EB buffer.  11. Save 1 ul of purified DNA for checking sonication at the Bioanalyzer (Agilent Technologies, G2939AA) in parallel with further processed samples. Libraries were generated using ThruPLEX DNA-seq 48S Kit (Rubicon Genomics, #R400427). Libraries were quantified by qPCR using the NEBNext Library quantification kit for Illumina (NEB, #E7630L). Libraries were diluted to 8 nM and pooled for multiplexing. The diluted concentrations and were quantified a second time using NEBNext kit. Libraries were denatured in 0.2 M NaOH and loaded into NextSeq 75-cycles High Output v2 Kit (Illumina, FC-404-2005) cartridge at a concentration of 2.5-3 pM. Sequencing runs were performed in an Illumina NextSeq 500 machine srHC-Seq
attributes.tag: GEO Accession
attributes.value: GSM3137743