GSM9408926: B cell, LPS activated, Replicate 2; Mus musculus; OTHER
2x106 cells were washed once in 5 ml L buffer (10 mM Tris HCl pH 7.5, 100 mM EDTA, 20 mM NaCl) and resuspended in 60 µl L buffer in a 2 ml tube. Samples were heated to 50°C for 2-3 min before addition of 40 µl molten CleanCut agarose (Bio-Rad 1703594), vortexing vigorously for 5 s before pipetting in plug mould (Bio-Rad 1703713) and solidifying 15-30 min at 4°C. Each plug was transferred to a 2 ml tube containing 500 µl digestion buffer (10 mM Tris HCl pH 7.5, 100 mM EDTA, 20 mM NaCl, 1% sodium N-lauroyl sarcosine, 0.1 mg/ml Proteinase K) and incubated overnight at 50°. Plugs were washed three times with 1 ml TE for 1-2 hours at room temperature with rocking, 10 mM PMSF was added to the second and third washes from 100 mM stock (Merck 93482). Plugs were then digested 1 h at 37°C with 1 µl 1000 U/ml RNase T1 (Thermo EN0541) in 200 µl TE. ½ an agarose plug of cells was equilibrated once in 100 µl 1x TdT buffer (NEB) for 30 min at room temperature, then incubated for 2 h at 37°C in 100 µl 1x TdT buffer containing 4 µl 10 mM ATP and 2 µl Terminal Transferase (NEB M0315L). Plugs were rinsed with 1 ml tris buffer (10 mM Tris HCl pH 8.0), equilibrated in 100 µl 1x T4 RNA ligase buffer (NEB) containing 40 µl 50% PEG 8000 for 1 hour at room temperature then incubated overnight at 25°C in 100 µl 1x T4 RNA ligase buffer (NEB) containing 40 µl 50% PEG 8000, 1 µl 10 pM/µl multiplexed TrAEL-seq adaptor 1 ([App]YYYYNNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGUAGCA/iBiodT/TGCUACACTCTTTCCCTACACGACGCTCTTCCG) and 1 µl T4 RNA ligase 2 truncated KQ (NEB M0373L). Plugs were then rinsed with 1 ml tris buffer, transferred to 15 ml tubes and washed three times in 10 ml tris buffer with rocking at room temperature for 1-2 hours each, then washed again overnight under the same conditions. Plugs were pooled, equilibrated for 15 min with 1 ml agarase buffer (10 mM Bis-Tris-HCl, 1 mM EDTA pH 6.5), then the supernatant removed and 50 µl agarase buffer added per plug. Plugs were melted for 20 min at 65°C, transferred for 5 min to a heating block pre-heated to 42°C, 1 µl per plug β-agarase (NEB M0392S) was added and mixed by flicking without allowing sample to cool, and incubation continued at 42°C for 1 h. DNA was ethanol precipitated with 25 µl 10 M NH4OAc, 1 µl GlycoBlue, 330 µl of ethanol per plug and resuspended in 10 µl 0.1xTE. 40 µl reaction mix containing 5µl Isothermal amplification buffer (NEB), 3 µl 100 mM MgSO4, 2µl 10 mM dNTPs and 1µl Bst 2 WarmStart DNA polymerase (NEB M0538S) was added and sample incubated 30 min at 65°C before precipitation with 12.5 µl 10 M NH4OAc, 1 µl GlycoBlue, 160 µl ethanol and re-dissolving pellet in 130 µl 1xTE. The DNA was transferred to an AFA microTUBE (Covaris 520045) and fragmented in a Covaris E220 using duty factor 10, PIP 175, Cycles 200, Temp 11, then transferred to a 1.5 ml tube containing 8 µl pre-washed Dynabeads MyOne streptavidin C1 beads (Thermo, 65001) re-suspended in 300 µl 2xTN (10 mM Tris pH 8, 2 M NaCl) along with 170 µl water (total volume 600 µl) and incubated 30 min at room temperature on a rotating wheel. Beads were washed once with 500 µl 5 mM Tris pH 8, 0.5 mM EDTA, 1 M NaCl, 5 min on wheel and once with 500 µl 0.1x TE, 5 min on wheel before re-suspension in 25 µl 0.1x TE. 3.5 μl NEBNext Ultra II End Prep buffer, 1 µl 1 ng/µl sonicated salmon sperm DNA (this is added as a carrier) and 1.5 μl NEBNext Ultra II End Prep enzyme were added and reaction incubated 30 min at room temperature and 30 min at 65°C. After cooling, 1.25 µl 10 pM/µl TrAEL-seq adaptor 2 ([Phos]GATCGGAAGAGCACACGTCTGAACTCCAGTCUUUUGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T ), 0.5 µl NEBNext ligation enhancer and 15 µl NEBNext Ultra II ligation mix were added and incubated 30 min at room temperature. The reaction mix was removed and discarded and beads were rinsed with 500 µl wash buffer (5 mM Tris pH 8, 0.5 mM EDTA, 1 M NaCl) then washed twice with 1 ml wash buffer for 10 min on wheel at room temperature and once for 10 min with 1 ml 0.1x TE. Libraries were eluted from beads with 11 µl 1x TE and 1.5 µl USER enzyme (NEB) for 15 min at 37°C, then again with 10.5 µl 1x TE and 1.5 µl USER enzyme (NEB) for 15 min at 37°, and the two eluates combined. An initial test amplification was used to determine the optimal cycle number for each library. For this, 1.25 μl library was amplified in 10 µl total volume with 0.4 µl each of the NEBNext Universal and any NEBNext Index primers with 5 µl NEBNext Ultra II Q5 PCR master mix. Cycling program: 98°C 30s then 18 cycles of [98°C 10 s, 65°C 75 s], 65°C 5 min. Test PCR was cleaned with 8 µl AMPure XP beads (Beckman A63881) and eluted with 2.5 µl 0.1x TE, of which 1 µl was examines on a Bioanalyser high sensitivity DNA chip (Agilent 5067-4626). Ideal cycle number should bring final library to final concentration of 1-3 nM, noting that the final library will be 3 cycles more concentrated than the test anyway. 21 µl of library was then amplified with 2 µl each of NEBNext Universal and chosen Index primer and 25 µl NEBNext Ultra II Q5 PCR master mix using same conditions as above for calculated cycle number. Amplified library was cleaned with 40 µl AMPure XP beads (Beckman A63881) and eluted with 26 µl 0.1x TE, then 25 µl of this was again purified with 20µl AMPure XP beads and eluted with 11µl 0.1x TE.