GSM6634268: HAP1_short-read_nucleoplasmfraction_rep2; Homo sapiens; RNA-Seq
For nuclear fractionation and RNA isolation, cell pellets were resuspended in Buffer A (10 mM HEPES•KOH pH 7.5, 10 mM KCl, 10% glycerol, 340 mM sucrose, 4mM MgCl2, 1 mM DTT, 0.1% Triton X-100, 1 x Protease Inhibitor Cocktail (PIC) [1mM AEBSF, 0.8 μM aprotinin, 20 μM leupeptin, 15 μM pepstatin A, 40 μM bestatin, 15 μM E-64; from 200x DMSO stock]) to obtain nuclei. Purified nuclei were extracted with 0.5 M Urea and 0.5% NP-40 substitute to solubilize loosely bound factors from chromatin and fractionated by centrifugation (Werner, 2015;Werner, 2017). The supernatant was termed as the nucleoplasm fraction and the pellet as the chromatin fraction. 1 μL of a 1:10 diluted ERCC RNA Spike-in standards aliquot (Life Technologies 4456740) was added to each nuclear fraction prior to addition of TRIzol reagent (Life Technologies 15596026). RNA from each fraction was processed using Zymo RNA Clean & Concentrator kit (Zymo Research R1017) with on-column DNase digestion. Ribosomal RNA was removed using RiboMinus kit (Invitrogen A15026) for HAP1 short- and long-read samples. For short-read RNA-seq, 100 ng rRNA-depleted RNA from each nuclear fraction was used with the NEBNext Ultra II Directional library kit (NEB E7765S) to prepare RNA-seq libraries. RNA-seq libraries of nuclear fractions (chromatin extract and nucleoplasm extract) from three independent cultures were sequenced on the Illumina HiSeq 4000 to obtain 50bp single end reads. For long-read RNA-seq, two HAP1 long-read sequencing libraries were generated as follows. rRNA-depleted RNA was polyadenylated with E. coli Poly(A) Polymerase (M0276S) (for later priming with long read sequencing primers) and precipitated with ethanol. Polyadenylated chromatin fraction RNA was subjected to library preparation according to the Oxford Nanopore direct cDNA sequencing kit protocol (SQK:DCS109). 10-50 fmol of library was loaded onto a MinION (MIN-101B) flow cell (R9.4.1; FLO-MIN106D) and run for 48 hours.