GSM5850496: chr-RNA #3; Mus musculus; OTHER
Chromatin-associated RNA (Chr-RNA) was isolated from ~20M cells as published previously (Henriques et al, Mol Cell 2013), but ﻿0.1% Triton-X-100 was added after pellets were resuspended in buffer B. Incubations were all performed on ice, 8 minutes in buffer A and two incubations in buffer B for 15 minutes. The washed chromatin pellets were resuspended in Trizol, RNA was extracted using Direct-zol RNA miniprep columns (Zymo Research), treated with RQ1 DNase (Promega), then cleaned up again using a total RNA purification kit (Norgen Biotek Corp). First, RNA was reverse transcribed with Superscript IV (Thermo Fisher), using a gene-specific primer in mKate2 annealed 221nt downstream of the variable regions. cDNA was RNase-treated and then concentrated using 2x RNA XP beads (Beckman Coulter) to limit the number of parallel PCR reactions that had to be set up. Sequencing libraries were generated through 17-20 PCR cycles using NEBNext Ultra II Q5 (New England Biolabs) and NEBNext Multiplex oligos, uniquely indexing each sample. Amplicons were cleaned up using 1.5x AMPure XP beads (Beckman Coulter) and concentrations were quantified using the NEBNext Library Quant Kit for Illumina, before mixing indexed samples and checking the size distribution by TapeStation (Agilent).