GSM8973688: 32_H1130_pro; Homo sapiens; RNA-Seq
Genomic DNA from cells (in 10M cell increments) were resuspended in 50 μl ChIP lysis buffer (1% SDS, 10 mM EDTA, in 50 mM Tris-HCl, pH 8.1) and pipetted up and down. Lysed cells were transfered to a 96-well plate or 8-well strip, then incubated at 65°C for 10 min. Then sample was cooled to 37°C, and 1 μl Rnase cocktail (Ambion, AM2286) was added, again mixed by pipetting and spun down followed by incubation at 37°C for 30 min. 5 μl proteinase K (NEB P8107) was added and the sample was mixed by pipetting. To isolate genomic DNA, we added 36.4μl Ampure XP to the sample, mixed thoroughly by pipetting, incubated for 5 min, and used a magnet to isolate genomic beads from the lysed sample. We pipetted off the supernatant and washed the sample 3 times with 80% ethanol while on the magnet. After drying the pellet for 5 min, gDNA was then eluted in 45 μl ddH2O, yielding on average 3-6 pg per cell. We then used 0.6 μg of genomic DNA from each sample for qPCR to determine optimal PCR cycle number for library preparation. Each 10 μL qPCR reaction contained 5 μL of NEBNext Q5 Hotstart HiFi PCR master mix (NEB # M0543L), FwdInnerSeq and RevInnerSeq at 500 nM each (final concentration; Supplementary Table 30), 0.6 μg of genomic DNA, 1.7 μL of SYBR (diluted 1/10000), and added water to have a final volume of 10 μL. Once the optimal PCR cycle was determined, 50 μL PCR reactions were used to amplify the amplicon, and the number of reactions were scaled to the total available gDNA. The PCR reactions from each sample were pooled, and 50 μL was taken for amplicon purification. Amplicons were purified using a two-step Ampure XP method. 0.65x Ampure XP was added to the sample, followed by mixing via pipet, and incubating for 5 min. The sample was applied to the magnet and the supernatant was isolated for further purification. To the supernatant, an additional 1.0x Ampure XP was added to the sample to capture PCR amplicon. The sample was incubated for 5 minutes, and then magnetic beads were captured by magnet. The supernatant was removed and discarded, and the captured beads were washed 3 times with 80% ethanol. The sample was dried for 5 min and eluted with 25 μL of H2O. Samples were analyzed on a TapeStation to assess amplicon purity and size estimation, and the concentration of amplicon DNA was measured using Qubit. Samples were pooled based on the concentration of the specific target amplicon percentage and sequenced on a Nextseq 2000 with custom read1 primer hU6_R1 and custom index primer sgPuro_I.