GSM4059913: Merged UMI-4C CXCL11 control; Homo sapiens; OTHER
Formaldehyde crosslinked human islets (estimated ~4 million individual cells) were treated with ice-cold lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% TX-100, 0.5% IGEPAL-CA-630, Sigma; 5mM EDTA, AppliChem; 1X protease inhibitor cocktail, Millipore) and permeabilized with SDS (Millipore) and TX-100. Nuclei were digested with DpnII (New England Biolabs) for 16h at 37ºC. Afterwards, more DpnII was added for 4h and ligated with T4 DNA ligase (Promega) at 16ºC overnight. Cross-link was reversed by overnight incubation with Proteinase K at 65°C. Next, after 30 min incubation with 30 µl of 10 mg/ml RNase A at 37°C, DNA was purified by phenol/chloroform and ethanol precipitation. DNA was then resuspended in 10 mM Tris-HCl pH 8 and 5 ug aliquots were sonicated by Covaris S2 to fragment sizes in the range of 450-550 bp. Sonicated DNA was next incubated for 30 min at 20ºC with 20 µl 10× end-repair buffer and 10 µl end-repair mix (NEB E6050S). The reaction was next cleared by 2× AmpureXP beads (Agencourt AMPure XP, Beckman Coulter) and eluted in 75 µl EB (10 mM Tris-HCl pH 8). A-tailing was performed on the elute by adding 10 µl NEB buffer 2, 4 µl Klenow fragment (NEB M0212L) and 10 µl 10 nM dATP 20 min at 37 ºC and 20 min at 75ºC to inactivate the reaction. DNA was next dephosphorylated with 2 µl calf intestinal alkaline phosphatase (NEB M0290S) and incubated at 50°C for 60 min and immediately purified by 2x AmpureXP beads. Illumina forked adaptors were added (final concentration 0.4 µM) and incubated with 80 µl quick ligase buffer and 5 µl of Quick Ligase (NEB M2200S) for 20 min at 25°C. DNA was next denatured at 96ºC for 5 min, moved to ice for 5 min and then purified by 0,8x AmpureXP beads to release the non-ligated strand of the adaptor. Denatured DNA was quantified by Qubit (ssDNA HS Assay, Q32851, Invitrogen). 200 ng of DNA were used for library preparation by nested PCR. The first PCR reaction was performed with 10 µl GoTaq Flexi Buffer, 3 µl MgCl2 25mM, 1 µl dNTP 10 mM, 2 µl US bait primer and 2 µl Illumina universal primer 10 µM (0,4 µM final concentration each primer) and 1 µl GoTaq (Promega, M5005) with the following program: 2 min 95°C, 20 cycles of 30 s 95°C, 30 s 56°C and 60 s 72°C and final extension of 5 min 72°C. After the first PCR reaction, the DNA was purified with 1× AmpureXP beads, and 31 µl of the elute was used to perform a second PCR reaction following the same conditions as the first one but using the DS bait primer and 17 amplification cycles instead of 20. Finally, DNA was purified by 0,8x AmpureXP beads. To increase molecular complexity, each library was obtained by pooling 5-10 PCRs per viewpoint. UMI-4C
GEO Accession
GSM4059913