title: GSM7136747: HEK293,H1.2KO,H1.3KO,H1.0KO,H1.5KO,H1.4KO; Homo sapiens; RNA-Seq
library_construction_protocol: For library generation, 1 x 106 cells were spiked with 5% permeabilizedDrosophilaS2 cells for data normalization and used as input for PRO-seq. Nascent RNA was labeled through a biotin-NTP run-on as follows: Permeabilized cells were added to an equal volume of a 2X run-on reaction mix. and incubated at 30 °C for 5 min. RNA was isolated using TRIzol Reagent. Isolated RNA was fragmented by base hydrolysis with 0.25 N NaOH for 9 min on ice, followed by neutralization with one volume of 1 M Tris-HCl, pH 6.8. To select for nascent RNA, 48 μL of washed Streptavidin M-280 magnetic beads in binding buffer were added to the fragmented RNA, and samples were rotated at room temperature for 20 min. The Streptavidin M-280 magnetic beads were washed twice in each of the following three buffers: high salt buffer , binding buffer, and low salt buffer. Beads were resuspended in TRIzol LS Reagent  and heated at 65 °C for 5 min, cooled to room temperature, and heated at 65 °C for 5 min again to elute the RNA from the beads. Nascent RNA was resuspended in 10 µM of the VRA3 3′ end adapter in T4 Ligase buffer and was 3′ end ligated with T4 RNA ligase I for 2 h at room temperature. The sample was enriched for ligated nascent RNAs by binding to Streptavidin M-280 magnetic beads as described above. The beads were washed twice in high, binding, and low salt buffers, then once in 1X ThermoPol Buffer. To prepare the nascent RNA for 5′ end adapter ligation, the 5′ ends of the RNA were decapped using RppH suspended in RppH buffer, then 5′ end was recapped with RNA 5′ pyrophosphohydrolase at 37 °C for 1 h in T4 ligase buffer. Beads were then washed in high salt buffer, low salt buffer, and 1X T4 PNK Reaction Buffer. Samples were treated with T4Poly-nucleotideKinase for 1 h at 37 °C, and the reverse 5′ RNA adapter VRA5 was ligated to the RNA by incubation with T4 RNA ligase I for 2 h at room temperature. Following the 5′ end ligation, beads were washed twice in high, binding, and low salt buffers, then once in 0.25X FSS Buffer . Reverse transcription was performed using Superscript IV Reverse Transcriptase with 25 pmol of the Illumina TRU-seqRP1Primer. The RT product was eluted from the beads by heating the samples twice at 95°C for 30 s. All libraries were amplified by 12 cycles of PCR with 12.5 pmol of Illumina TRU-seq RPI-index primers, excess RP1 primer, and Q5 DNA Polymerase. The samples were sequenced using a HiSeq instrument with a mid-output 150bp cycle run. Six nucleotide Barcode specific primers were used in order to isolate the results from each of the specific samples (WT, 1-KO, 2-KO, 4-KO and 5-KO).